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Placenta 30 (2009) A.1–A.111

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Placenta

journal homepage: www.elsevier .com/locate/placenta

Abstracts for the Forthcoming International Federation of PlacentaAssociations Meeting 2009

Abstract Outline - IFPA 2009

Final Keynote Symposium N1 Abstracts (K1–K2) A.2

Symposium 1: Sex and the Placenta (S1–S3) A.3–A.4

Symposium 2: Prediction of Adverse Pregnancy Outcome (S4–S6) A.4–A.5

Symposium 3: IUGR, the Placenta and Programming (S7–S9) A.5–A.6

Symposium 4: Trophoblast and Endometrial Interactions (S10–S12) A.7–A.8

NIH Senior Researcher Award A.8

New Investigator Oral Session 1 (N1–N6) A.8–A.11

New Investigator Oral Session 2 (N7–N12) A.11–A.14

Trophoblast Research Award (TR1) A.14

IFPA Award in Placentology (L2–L3) A.15

Poster Abstracts (P01.01–P19.08) A.16–A.111

doi:10.1016/j.placenta.2009.08.001

Abstracts / Placenta 30 (2009) A.1–A.111A.2

K1.MARSUPIALS: PLACENTAL MAMMALS WITH A DIFFERENCE

Marilyn B. Renfree, Department of Zoology, The University of Melbourne,Victoria 3010, Australia

Viviparity is widespread in the animal kingdom and made possible by thedevelopment of a placenta. The placenta is the most varied organ withinthe Mammalia, but the term placental mammal (aka eutherian mammal)gives the impression that the placenta is found only in that infraclass.There are three major groups of mammals, the egg-laying monotremesand the viviparous marsupials and eutherians. Whilst the eutherianplacenta is well understood, the marsupial placenta is often ignoreddespite the recognition of it by the two great placentologists of the lastcentury, Harland Mossman and Emmanuel C. Amoroso. Both understoodthat marsupials had a fully functional placenta and emphasized the valueof comparative placental physiology.There are many similarities, as well as some differences, in the marsupialembryo and its fetal membranes. In marsupials, the yolk sac forms thedefinitive chorio-vitelline placenta. Only very few marsupials, such asthe bandicoot, have a chorio-allantoic placenta, which supplements theplacental functions of the yolk sac. The yolk sac consists of two parts:a bilaminar, non-vascular region (trophoblast and yolk sac endoderm), anda trilaminar, vascular part (trophoblast, yolk sac endoderm and meso-derm). The bilaminar placenta appears to serve primarily in the uptake ofuterine secretions, and the trilaminar part may be more important for gasexchange. The degree of this functional differentiation within the yolk sacplacenta differs between species in the relative surface area that isattached to the endometrium, in trophoblast thickness, in yolk sac fusionwith the luminal epithelium and most markedly in the degree ofinvasiveness.Implantation, the transition from free floating blastocyst to the implantedembryo, is a critical point in mammalian pregnancy. The extent ofimplantation and the degree to which the blastocyst becomes closelyassociated with the maternal endometrial tissues dictates the type ofplacenta developed. In species in which the blastocyst expands beforeattachment such as rabbit, dog, ferret and many marsupials, a large area oftrophoblast is exposed to the uterine lumen. In the pig, horse, kangarooand wallaby there are no such special areas and superficial attachmentoccurs at the unspecialized endometrial surfaces over the entire tropho-blastic area.In marsupials, placental physiology has only been extensively studied inthe tammar wallaby. Despite the lack of invasion, in the tammar there isnevertheless maternal recognition of pregnancy in response to trophoblastformation. Contrary to popular opinion, the tammar placenta also elabo-rates hormones: at term it secretes prostaglandin F2a and cortisol, onlyincipient amounts of progesterone and oestrogen, and it expresses thegenes for growth hormone, IGF2, relaxin, prolactin and luteinizinghormone b. As in eutherian mammals, there is genomic imprintingimportant for placental function.Despite the relatively short gestation and short period of placentation, it isclear that the trophoblast is as important for successful pregnancy in themarsupial as it is in eutherian mammals. Marsupials are certainly placentalmammals. However marsupials have an additional trick in their pouches,with the physiologically sophisticated and extended lactation that hasallowed them to exchange the umbilical cord for the teat!

K2.EVOLUTION OF EPIGENETIC SILENCING IN MAMMALS

Jennifer A. Marshall Graves, Research School of Biological Science,Australian National University, Canberra, Australia; Email:[email protected]

In mammals, several clusters of genes display parent-specific silencing(genomic imprinting), and one whole X chromosome is silenced in somatictissue of females. To understand how these silencing mechanisms evolvedand provide clues to how they work, we have investigated the origin andevolution of imprinting and X inactivation by examining orthologousregions in distantly related mammals (marsupials and the egg-layingmonotremes).X chromosome inactivation in females is the premier example of epige-netic silencing. In placental mammals, XCI involves DNA methylation andbinding with variant and modified histone, under the control of the XISTgene. The marsupial X, equivalent to the ancient region of the human andmouse X, is inactivated in marsupials, but the phenotype and molecularmechanism is very different. Silencing occurs only for the paternal X, and istissue-specific and incomplete to different extents for different genes. Itappears to be a stochastic process, in which alleles of each gene havea certain probability of expression, rather than being expressed at anintermediate level. In monotremes, as in birds, genes on the multiple Xchromosomes are partially dosage compensated, again by a stochasticprocess. This suggests evolution from monoallelic expression of autosomalloci.The molecular mechanism of XCI appears to be quite different in marsu-pials. XIST is absent from the marsupial genome, no DNA methylationdifferences are detectable between active and inactive X chromosomes,and immunofluoresence shows that although active histone marks areabsent from the inactive X (as for placental mammals), inactive marks donot accumulate. This enables us to propose an evolutionary pathway forbuilding up a complex silencing pathway.Genomic imprinting occurs in marsupials as well as placental mammals,but not in monotremes or birds. Thus the first appearance of imprintingcoincides with the evolution of viviparity and placentation. We haveidentified how imprinted domains (including XIST) were put together fromnon-imprinted components. Again, ancient mechanisms to control geneexpression via epigenetic silencing may have been recruited into thecomplex imprinting and inactivation systems.Imprinted genes are disproportionately involved in human diseasebecause a mutant allele has no back-up. Diseases involving maternal-fetalinteractions might be particularly likely to involve imprinted genes, as Isuggested 10 years ago for pre-eclampsia.

Abstracts / Placenta 30 (2009) A.1–A.111 A.3

Symposium 1: Sex and the Placenta

S1.SEX AND THE HUMAN PLACENTA

VL Clifton*, A Osei-Kumah, NA Hodyl, N Scott and MJ Stark, RobinsonInstitute, University of Adelaide, Adelaide

The placenta plays a central role in the development of the fetus bymodulating the supply of nutrients and oxygen throughout pregnancy. Wehave identified that the placenta adapts to the presence of a maternalpathophysiology in a sexually dimorphic manner which results in differ-ences in fetal growth. In pregnancies complicated by asthma or mildpreeclampsia we have reported the female fetus reduces her growth inresponse to these conditions which ensures her survival in the presence ofanother adverse event that may compromise nutrient or oxygen supply.Conversely, the male fetus continues to grow normally in the presence ofmaternal asthma or mild preeclampsia but is at risk of a poor outcome inthe presence of a second stress. We propose that the sexually dimorphicresponse of the fetus is derived from differences in placental adaptation toa pathophysiological condition. In the presence of a female fetus andmaternal asthma, we have observed global gene changes in the placentaaccompanied by significant alterations in microRNA expression. Down-stream of these alterations we have observed differences in proteinexpression especially in relation to placental cytokines and the glucocor-ticoid receptor. In the presence of a male fetus there are fewer changes inglobal placental gene expression and fewer microRNA alterations and wehave observed no alterations in placental cytokine expression or gluco-corticoid receptor expression. These differential adaptations ensureincreased survival of the female fetus and continued growth of the malefetus in adverse conditions.

S2.GLUCOCORTICOIDS AND DRUG TRANSPORTERS: FROM PLACENTA TOFETAL BRIAN

Stephen G. Matthews1, Sophie Petropoulos1, William Gibb2, 1Departmentsof Physiology, Obstetrics and Gynecology and Medicine, University ofToronto, Canada. 2 Departments of Obstetrics and Gynecology and Cellularand Molecular Medicine, University of Ottawa, Canada

The fetus may be exposed to a number of therapeutic and non-therapeuticdrugs, environmental toxins and other chemicals during pregnancy. Lip-ohilic compounds can rapidly transfer from the maternal to the fetalcompartment to affect developing organs. The fetus is protected froma number of these factors through the expression of drug transporters inthe placenta and fetal organs. Multidrug-resistance P-glycoprotein (P-gp),encoded by the ABCB1 gene, and breast cancer resistance protein (BCRP),encoded by the ABCG2 gene, are ATP-dependant extrusion pumps. Theyhave a broad spectrum of substrates that include endogenous factors /hormones (i.e. glucocorticoids), anti-cancer drugs, anti-HIV retroviraldrugs as well as herbicides and pesticides. We and others have shown thatthe effects of these drugs on fetal development are sex-specific and thatthere is some sex-specificity in the transfer of these substrates from themother to the fetus. Since one of the primary sites of teratogenesis in thefetus is the brain, we have investigated the expression and regulation ofthese drug transporters in the placenta and the fetal blood bran barrier, inboth male and female fetuses. There are dramatic changes in the expres-sion of these transporters over the second half of gestation in the mouse,guinea-pig and human placenta and fetal blood-brain barrier. While thereare no major sex differences in the developmental profile of these drugtransporters, there do appear to be sex differences in their regulation bysteroids. Defining the processes that regulate the expression of P-gp andBCRP, and therefore drug transport between the mother and fetus, areimportant in the development of maternal and fetal therapies. Further,determining whether this regulation is specific to the sex of the fetus iscritical.Supported by: Canadian Institutes of Health Research (CIHR)


S3.SEX, DRUGS AND PROGRAMMING OF THE KIDNEY AND PLACENTA

Karen Moritz, School of Biomedical Sciences, University of Queensland,Australia

The ‘‘Developmental Origins of Health and Disease’’ hypothesis has causedrenewed interest in understanding the factors regulating fetal develop-ment. A multitude of prenatal perturbations have now been identified ascontributing to the onset of many adults diseases including cardiovascular,metabolic and renal disease. An interesting feature in many animal modelsof developmental programming is the disparity between males andfemales in the onset and severity of disease outcomes. Using a variety ofanimal models (maternal glucocorticoid exposure, maternal low proteindiet, uteroplacental insufficiency, prenatal alcohol exposure), our studieshave identified alterations in kidney development as being a commonfeature of many of these models. The formation of a low nephronendowment may result in impaired renal function which in turn mayunderlie the observed disease outcome. However, the same prenatal insultdoes not always affect males and females to the same degree. For example,the degree of nephron deficit and glomerular enlargement in males andfemales may not be the same despite a similar insult. Even if the deficitsare similar between the sexes, this does not always result in a similardegree of hypertension or impairments in renal function. Whilst levels ofsex hormones after birth are undoubtedly important, there is now growingevidence that the placenta from males and females differ in theirresponses to insult and this plays a crucial role in regulating fetal growthand development.Most recently, using rodent models, our studies have focused on themolecular changes induced in the placenta and fetal kidney followingexposure to short term elevations in maternal glucocorticoids or low dosesof ethanol throughout pregnancy. We have shown that changes inexpression of receptors of the renin-angiotensin system along with genesinvolved in the process of branching morphogenesis and fluid balanceoccur in the developing kidney and placenta. These are often sex specificwhich may in part explain the observed sex differences in diseaseoutcomes.

Symposium 2: Prediction of Adverse Pregnancy Outcome

S4.MATERNAL PLASMA NUCLEIC ACIDS AS A TOOL FOR PRENATALDIAGNOSIS AND MONITORING

Dennis Lo, The Chinese University of Hong Kong, Hong Kong

The discovery of cell-free fetal nucleic acids in maternal plasma in 1997 hasopened up new possibilities for non-invasive diagnosis and monitoring. Ithas been demonstrated by many groups that the concentrations of circu-lating fetal nucleic acids are elevated in a number of pregnancy-associatedpathologies, most notably preeclampsia. Early work in this area hadfocused on the use of Y chromosomal markers, which could only be used ifthe fetus was male. More recent work has resulted in a number of sex- andpolymorphism-independent fetal nucleic acid markers, including DNAmethylation markers, mRNA and miRNA markers. In addition, exciting newadvances in analytical technologies for circulating fetal nucleic acids havebeen made. One of these involves the use of single molecule countingapproaches such as digital PCR and next-generation DNA sequencing.These single molecule approaches offer unprecedented precision in thequantitative analysis of circulating fetal nucleic acids and will be expectedto accelerate the clinical applications in this field.

S5.ONE-CARBON METABOLISM GENETIC POLYMORPHISMS AND ADVERSEPREGNANCY OUTCOMES

Denise Furness, Gus Dekker, Dee McCormack, Rachael Nowak, StevenThompson, Claire Roberts. University of Adelaide, Australia

Introduction: The aim of this project was to test genetic polymorphismsinvolved in one-carbon metabolism and vitamin-B12 transport forpotential associations with adverse pregnancy outcomes.Methods: In a prospective observational study, DNA was obtained from586 nulliparous Caucasian couples without fertility treatment. DNA wasgenotyped for MTHFR C677T, MTHFR A1298C, MTR A2756G, MTRR A66G,MTHFD1 G1958A and TCN2 C766G polymorphisms. Chi-square analysiswas used to compare genotype frequencies with pregnancy outcomes.Pregnancies were classified as healthy (n¼261), preeclampsia (PE, n¼38),gestational hypertension (GHT, n¼32), small-for-gestational-age (SGA,n¼60) and PE+SGA (n¼22). Associations between maternal, paternal andneonatal genotypes with customised birthweight centiles and placentalweight were determined using ANOVA with SIDAK post-hoc analyses.Results: All genotypes were in Hardy-Weinberg equilibrium. The maternalMTR 2756 G allele was associated with decreased placental weight (-87g,P¼0.040). Both paternal and neonatal MTR 2756 G alleles were associatedwith lower birthweight (-12%, P¼0.028 and -10%, P¼0.039, respectively)while the latter was also associated with PE+SGA (P >0.000). NeonatalMTRR GG genotype was associated with GHT and PE with SGA (P¼0.033,P¼0.011). Neonatal MTHFD1 GG genotype was twice as frequent in PE andGHT (P¼0.037; P¼0.019) and neonatal TCN2 GG genotype doubled in SGA(P¼0.042) compared with healthy pregnancies.Discussion: Our study shows that MTR A2756G, MTHFD1 G1958A andTCN2 polymorphisms are related to adverse pregnancy outcomes. MTRwith vitamin-B12 as a cofactor, catalyses the methylation of homocysteineto methionine. Formation of methionine through this pathway representsan important component for synthesis of phospholipids, proteins, myelin,catecholamines, DNA, RNA and S-adenosyl methionine. TCN2 encodes thevitamin-B12 transport protein and MTHFD1 catalyses the conversion ofone-carbon derivatives of tetrahydrofolate, which are substrates formethionine, thymine and purine synthesis which are all important forhealthy placental and fetal development. Future studies will investigatethe role of these polymorphisms in the placenta.


S6.LOW BIRTHWEIGHT IS PRECEDED BY ALTERED IMMUNE CYTOKINEPROFILES ACROSS VERY EARLY PREGNANCY

S. Tong*1,2, Y. Sucintri Thio1, M. Permezel1, C. Russell1, H. Georgiou1.1University Department of Obstetrics and Gynaecology, Mercy Hospital forWomen, Australia. 2Centre for Women’s Health Research, Monash Instituteof Medical Research, Australia

Introduction: It is hypothesised that errors in immune-led implantationcause major diseases of pregnancy, notably low birthweight andpreeclampsia. Evidence for this comes from in vitro human (Hiby et al.J Exp Med 2004) and in vivo animal experiments (Hanna et al Nature Med2006). Critically, there is no clinical evidence temporally linking the immunesystem around the time of implantation with the development of seriousdiseases of later pregnancy.We looked for evidence of differential immune system profiles at earlypregnancy preceding birth of a small for gestational age (SGA; birthweight<10th centile).Methods: We prospectively collected maternal serum at 7-11 weeks’gestation. After recruiting 756 participants, we selected 60 cases where thedelivery outcome was a SGA baby and 71 controls (median birthweight50th centile [range 30th-80th]). We used a multiplex protein bead array toquantify 27 pro and anti-inflammatory cytokines, chemokines and growthfactors.Results: Pregnancies complicated by SGA were preceded by profoundderangements in cytokine profiles. Of 21 detectable cytokines/chemo-kines, 14 varied significantly (p�0.004 all comparisons) among thosedestined to birth a SGA baby 30 weeks later, compared to controls. Besidesinterferon-g (IFN-g) which was raised, all other inflammatory cytokinesthat varied decreased with SGA, including major pro (Interleukin (IL)-2, IL-7, IL-12p70) and anti-inflammatory (IL-1 receptor antagonist, IL-4, IL-10,IL-13) cytokines.Combining four cytokines/chemokines (IFN-g, IL-7, IL-1 receptor antago-nist, and Eotaxin) predicts the occurrence of an SGA baby with 60%sensitivity, 90% specificity and a negative predictive value of 95%.Conclusion: The immune system may be involved in pathogenic mecha-nisms occurring as early as the middle of the first trimester causing at leasthalf of SGA deliveries.Early pregnancy immunodulation might be a novel therapeutic approachto decrease the immediate and lifelong health burden that affects babiesborn at low birthweight.

Symposium 3: IUGR, the placenta and programming

S7.REACTIVE OXYGEN AND NITROGEN SPECIES AND FUNCTIONALADAPTATION OF THE PLACENTA

Leslie Myatt, Center for Pregnancy and Newborn Research, University ofTexas Health Science Center San Antonio, USA

The type and amount of nutrients transferred, together with the type,intensity and timing of the hormonal signals it generates to influence bothmaternal and fetal systems, allows the placenta to control fetal growth anddevelopment. The placenta is in a constant state of growth and differen-tiation throughout gestation and changes its functional capacity byvascular growth and by trophoblast proliferation and differentiation. Boththe vasculature and trophoblast produce a variety of reactive oxygen andnitrogen species including superoxide and nitric oxide that regulate theirfunction. In addition interaction of NO and superoxide may producea more potent powerful pro-oxidant, peroxynitrite. Production of thesemediators, and hence placental patterns of growth and differentiation,may be altered by exposure to varying oxygen concentrations ranging fromhypoxia to hyperoxia. The high metabolic demands of the placenta meanthat pregnancy per se is a state of oxidative stress. This is further exacer-bated in conditions such a preeclampsia and IUGR or with obesity ordiabetes where many markers of oxidative stress and inflammation areincreased. The reactive oxygen and nitrogen species per se have effects assecond messengers in the placenta, but may also regulate activity of othermolecules, including proteins, lipids and DNA, via covalent modification.Superoxide produced from mitcochondria, and from enzymes includingxanthine oxidase and NADPH oxidase can give carbonylation of lysine,arginine, proline and threonine residues on placental proteins which willalter function. Peroxynitrite may cause nitration of specific tyrosine resi-dues in proteins, a covalent modification that can cause either, loss of, gainof or no change in protein function, Hence the extent of oxidative andnitrative stress at the tissue or cellular level may affect placental functionand hence fetal growth and development by covalent modification ofenzymes, receptors transporters and structural proteins.


S8.PLACENTAL PROGRAMMING OF METABOLIC HEALTH OF PROGENY

Julie A Owens, Kathy Gatford, Miles DeBlasio, Jeffrey Robinson, School ofPaediatrics and Reproductive Health, Faculty of Health Sciences andResearch Centre for Early Origins of Health and Disease, The RobinsonInstitute, University of Adelaide, Adelaide, SA, Australia

Placental insufficiency is a common pregnancy complication and majorcause of fetal growth restriction and reduced size at birth for gestationalage. Low birth weight consistently predicts an increased risk of adultdiabetes, accounting for a substantial proportion of its population preva-lence. In humans, this appears to be mediated through early onset defectsin insulin secretion, which then combines with onset of peripheral insulinresistance in early adult life to initiate progression of impaired glucosetolerance and diabetes. Experimental placental restriction has beeninduced in sheep and rodents and the life course and tissue/cellular andmolecular basis of its programming of insulin action and diabetes moredirectly characterised. In both species, placental restriction induces earlyonset defects in insulin secretion, followed by later onset of insulin resis-tance in progeny. In sheep, placental restriction impairs insulin secretionfrom before birth, in part due to reduced b cell mass. This persists intoadult life, despite increased pancreatic expression of PDX-1 and otherfactors that help restore b cell mass after birth, with impairment ofintrinsic b cell function, in association with reduced expression of calciumchannels a major and early defect following placental restriction. Placentalrestriction in sheep also does not impair insulin sensitivity before birth,with whole body insulin resistance only evident by one month of age,following catch-up growth and in association with central obesity. As inyoung human adults of low birth weight, this occurs with reduced skeletalmuscle expression of key insulin signalling molecules, including that ofIRS-1, subunits of PI3kianse, AKT and targets such as GLUT4. Therefore thetiming of onset of placentally programmed defects in insulin secretion andsensitivity varies, with molecular targets and windows of opportunity forintervention now being identified.

S9.ADAPTATION IN PLACENTAL NUTRIENT SUPPLY TO MEET FETALGROWTH DEMAND: IMPLICATIONS FOR PROGRAMMING

Colin P. Sibley, University of Manchester, UK

Fetal growth is absolutely dependent on supply of nutrients from mothervia the placenta. A variety of human and mouse data together suggest nowthat placental capacity to supply nutrient is adaptable in relation to fetaldemand. (1) In normal human pregnancy the activity, per mg membraneprotein, of the System A amino acid transporter in the microvillous plasmamembrane (MVM) of the placental syncytiotrophoblast is inversely relatedto the birthweight and size of baby. (2) In mice with a deletion of theplacental transcript of the insulin-like growth factor 2 (igf2) gene,placental weight is reduced compared to wild type mice at e16 (term e20)but fetal weight is not reduced at this time, perhaps because System Aexpression and activity, per g placenta, is higher in the knockout concep-tuses. (3) In the same knockout mice fetal calcium accretion is reducedcompared to wild type at e17, but returns to normal by e19 apparentlythrough an increased activity/expression of placental calcium transportmechanisms. (4) Coan et al (J. Physiol 586, 4567-4576, 2008) examinednatural inter-litter variation in placental transfer capacity in normal mice;they found that there are gestation dependent morphological and func-tional adaptations in the smallest placentas enabling them to maintainfetal size as compared to larger placentas. The variable nature of theplacental transfer adaptations in different situations, as found in thesedifferent studies, will variably affect the mix of nutrient transferred and sopotentially variably programme fetal homeostatic mechanisms.

Grant support from Tommy’s [Let’s Talk Baby] Charity, The Medical ResearchCouncil and The Wellcome Trust is gratefully acknowledged, as are thecontributions of many colleagues in the Manchester Maternal and FetalHealth Research Group.


Symposium 4: Trophoblast and endometrial interactions

S10.A FUNCTIONAL ROLE FOR UTERINE NATURAL KILLER (uNK) CELLS INEARLY PREGNANCY HUMAN DECIDUAS

Gendie Lash, University of Newcastle upon Tyne, UK

Uterine natural killer (uNK) cells are the major leucocyte in early preg-nancy decidua, comprising 70% of decidual leucocytes and 20-30% of alldecidual cells. They are phenotypically distinct from pheripheral blood NKcells in that they are CD56+CD16-. They have been proposed to playa crucial role in early pregnancy development, although this role is onlyjust becoming clear. Using a well characterised matrigel invasion assaywith placental vellous explants and uNK cell supernatants taken fromeither 8-10 or 12-14 weeks gestational age we have demonstrated thatonly uNK supernatants from 12-14 weeks gestational age has a stimulatoryeffect on EVT invasion. In contrast, total decidual cell isolates supernatantsfrom both 8-10 and 12-14 weeks gestational age stimulate EVT invasion. Inaddition, the effect of uNK supernatants from 12-14 weeks gestational agecould be partially blocked by neutralisng antibody to IL-8, an abundantcytokine whose production increases from 8-10 to 12-14 weeks gestationalage. The uNK supernatant effect on EVT invasion as associated with anincrease in protease activity and a decrease in EVT apoptosis. EVT arenaturally highly invasive although their invasive ability decreased after 10weeks gestational age. Our results suggest that later in gestation when EVTinvasion is still required, but to a lesser degree than in very early pregnancyuNK cells aid in facilitating this process.One of the other roles proposed for uNK cells in early pregnancy is inmediating the non-trophoblast component of spiral artery remodelling.Early in the remodelling process, in the absence of EVT, there is separationof the vascular smooth muscle cells, dilation of the vessel and somevascular permeability. uNK cells are a major source of several keyangiognic growth factors, production of which decreases with increasinggestational age, that may play a role in these early stages of remodelling. Inaddition, in early pregnant decidua uNK cells are localised around spiralarteries. Using a chorionic plate artery model we have shown that uNKsupernantants can facilitate similar precessess in vitro. The exact mecha-nisms of these actions are currently under investigation in our laboratory.Overall, we would suggest that uNK cells are involved in at least two keyprocesses of early pregnancy that change with gestational age. Firstly, inearly spiral artery remodelling and ten in mediating EVT invasion. Thetrigger for this switch in function is unclear. It is interesting to note thatearly in pregnancy uNK cells are a major producer of angiogenic growthfactors whose production decreases with increasing gestational agewhereas in contrast the uNK cell production of cytokines increases withgestational age.

S11.TROPHOBLAST-VASCULAR CELL INTERACTIONS IN EARLY PREGNANCY:HOW TO REMODEL A UTERINE ARTERY

Lynda Harris, University of Manchester, UK

During the first twenty weeks of pregnancy, extravillous trophoblasts(EVT) colonise the decidua and remodel the uterine spiral arteries as far asthe first third of the myometrium. This process leads to an irreversiblevasodilatation, ensuring that maximal blood flow is delivered to thematerno-fetal interface at an optimal velocity for nutrient exchange. Thereis accumulating evidence that subtle changes in vascular structure precedeEVT colonisation; however, full physiological transformation is only ach-ieved in the presence of trophoblast and requires vascular cell loss andextracellular matrix breakdown. Following extensive analysis of decidualspiral arteries between 8 and 12 weeks of gestation, we have observed thatendothelial cell hypertrophy and SMC disorganisation occur in the absenceof trophoblast, and instead correlate with the perivascular and intramuralaccumulation of uterine natural killer (uNK) cells. We hypothesise thatthese initial changes in vascular structure aid trophoblast entry into thearterial wall.Using a combination of monolayer co-cultures, immunohistochemicalanalysis of first trimester decidua and excised spiral arteries perfused withfirst trimester trophoblasts, we have shown that trophoblasts have thecapacity to induce vascular cell apoptosis, mediate elastin breakdown andupregulate protease expression in vascular smooth muscle cells (SMC).Trophoblasts express and secrete the apoptotic cytokine Fas ligand, andinduce apoptosis of vascular endothelial cells and SMC in vitro. A Fas ligandfunction-blocking antibody significantly inhibited apoptosis when addedto co-cultures, and reduced cleaved PARP expression and terminal deox-ynucleotidyl transferase dUTP nick end labelling (TUNEL) in spiral arterysegments perfused with trophoblasts or trophoblast-conditioned medium.EVT also express surface-associated TNF-alpha-related apoptosis-inducingligand (TRAIL), which can induce apoptosis of spiral artery SMC via ligationof TRAIL receptor-1 or -2. This mechanism requires intercellular contact,reinforcing the importance of intramural incorporation of trophoblast inthe remodelling process.In order to effect a permanent increase in the calibre of the spiral arteries,the internal elastic lamina and medial elastin fibres must be degraded. Wehave shown that first trimester trophoblasts utilise matrix metal-loproteinases (MMP) to mediate elastin breakdown, and that a specificMMP-12 inhibitor reduces elastase activity by >50%. Furthermore,vascular SMC also exhibit elastase activity, and spiral artery SMC upregu-late MMP-12 expression in response to trophoblast-derived factors.These data support a model in which the actions of uNK cells facilitatecolonization of the spiral arteries by EVT. SMC and EVT act cooperatively toeffect local degradation of elastin and other matrix components, whilsta subset of SMC respond to trophoblast with an elevated rate of apoptosis.These findings illuminate the complex steps involved in achieving vasculartransformation while maintaining vascular integrity in vivo.


S12.CYTOKINES REGULATE TROPHOBLAST-ENDOMETRIAL INTERACTIONSIN THE ESTABLISHMENT OF PREGNANCY

Eva Dimitriadis, Prince Henry’s Institute of Medical Research, Australia

Embryo implantation involves the initial adhesion of the trophectoderm tothe endometrial surface epithelium followed by the migration and inva-sion of the trophoblast through the maternal decidua remodeling theendometrial arteries to sequester blood to the developing placenta. Thesehighly regulated processes are critical for pregnancy success – inadequa-cies in which can have far reaching consequences including early miscar-riage, preeclampsia, preterm birth and intrauterine growth restriction. It islikely that abnormalities in the very early stages of trophoblast invasioncontribute to the development of such pregnancy complications. Due tothe great difficulty in obtaining very early implantation sites very little isknown about how the trophoblast interacts with the decidua to facilitatethe process of trophoblast invasion. Numerous cytokines are produced atthe human maternal-fetal interface including interleukin (IL) 11, leukemiainhibitory factor (LIF) and activin A. IL-11 and LIF, two IL6 family membercytokines appear to have distinct roles in trophoblast invasion shownusing both ex vivo and in vitro models. The TGF beta superfamily membercytokine, activin A similarly regulates trophoblast function. The role,interactions and mechanisms of action of these cytokines will be pre-sented. The data suggests that these locally produced cytokines haveimportant roles in the very early stages of placentation.

NIH Senior Researcher Award

THE PLACENTA IS A PROGRAMMING AGENT FOR CARDIOVASCULARDISEASE

K. Thornburg1,2,3, P.F. O’Tierney1 & S. Louey1,2. Heart Research Center1,Departments of Medicine (Cardiovascular Medicine)2, and Physiology andPharmacology3, Oregon Health and Science University, USA.

Cardiovascular disease remains the number one killer in western nationsin spite of declines in death rates following improvements in clinical care;disease rates are rapidly increasing in developing countries. Cardiac deathsare primarily due to coronary artery occlusion, progressive heart failure orsudden death from fibrillation. It has been 20 years since David Barker andcolleagues showed that slow rates of prenatal growth predict mortalitydue to ischemic heart disease. Thus, undoubtedly fetal undergrowth andits associated cardiovascular diseases are due to placental inadequacies.This conclusion is supported by a number of studies linking placentalcharacteristics with various adult diseases. Martyn and colleagues’discovered a ‘‘U’’ shaped relationship between placental-to-fetal weightratio and heart disease. This is powerful evidence that placental growthregulating processes initiate vulnerabilities for later heart disease inoffspring. Recent unpublished evidence from Finland indicates thatplacental morphological characteristics predict risks for coronary arterydisease, heart failure, hypertension and several cancers. The level of riskimparted by placental shape is sex dependent. Further, maternal diet andbody composition strongly influence placental growth, levels of inflam-mation, nutrient transport capacity and oxidative stress with subsequenteffects on offspring health. We have used several animal models todetermine the placental roots of vulnerability for heart disease. Drs. Shautand Stadler have shown that abnormal endothelial development in theplacenta is associated with undergrown myocardial walls in the embryo.As shown in our laboratory and several others, placental insufficiencyleads to depressed maturation and proliferation of working myocytes inthe fetal sheep heart. Together these models suggest that the ultimatefitness of the heart for a life of hard work is determined by hemodynamic,growth factor, and oxygen/nutrient cues before birth, all of which areinfluenced, if not regulated by the placenta.

New Investigator Oral Session 1

N1.MATERNAL DIETS HIGH IN SUGAR AND FAT IMPAIR LABYRINTHINEDEVELOPMENT AND AMINO ACID TRANSFER IN THE MOUSE PLACENTA

Amanda N Sferruzzi-Perri, Owen R Vaughan, Graham J Burton, Abigail LFowden, Centre for Trophoblast Research, University of Cambridge, UK

Women of reproductive age are consuming increasing amounts of dietarysugar and fat. In mice, high sugar-high fat (HSHF) diets during pregnancyhave adverse consequences postnatally but little is known about theireffects in utero. This study compared fetal and placental growth in mice feddiets of differing fat and sugar content.Methods: C57BL6/J female mice were fed diets of natural plant material(N, energy content from fat 11%, sugar 3%) or of processed ingredients highin sugar and animal fat, HSHF1 (energy; fat 22%, sugar 20%) or HSHF2(energy; fat 30%, sugar 36%) throughout pregnancy. On day (D) 19 ofpregnancy, placental transport of 14C-methyl aminoisobutyric acid(MeAIB) was measured in vivo under anaesthesia, and placental histologyassessed. Differences between diets were determined by linear mixedmodel repeated measures ANOVA with Sidak post hoc test and consideredsignificant if P<0.05.Results: Maternal energy intake per day remained constant irrespective ofdiet. There was no effect of diet on litter size or viability. Fetal weight wassimilar while placental weight was 11% lower on both HSHF than N diets(Figure). The fetal:placental weight ratio was 10-14% higher in HSHFrelative to the N groups (P<0.001). Compared to the N group, labyrinthinevolume was reduced by 12-16% on the HSHF diets (P<0.04). These changeswere accompanied by a 25-35% reduction in placental MeAIB transfer inmothers fed the HSHF diets (Figure).

Figure. Effect of maternal High sugar-high fat (HSHF) diets on feto-placental growthand placental transport.

Conclusions: The source of energy in the maternal diet has an importantrole in feto-placental development. Feeding HSHF diets during mousepregnancy reduced placental growth and amino acid transport withoutaffecting fetal growth near term. Other compensatory mechanisms musttherefore exist to maintain fetal growth in response to HSHF diets. Theefficiency of other nutrient transfer systems in the small HSHF placentaand maternal metabolic profiles, are currently being investigated.


N2.ACTIVIN A - AN EARLY INDUCER OF DECIDUALIZATION - SYNERGISESWITH IL11 TO PROMOTE PROGESTERONE INDUCED DECIDUALIZATION

Ellen Menkhorst*, Lois A. Salamonsen, Jin Zhang, Craig A. Harrison, Jun Guand Evdokia Dimitriadis, Prince Henry’s Institute, Australia

Decidualization of endometrial stromal cells (ESC) is a critical requirementfor the formation of a functional placenta. The mechanisms of human(H)ESC decidualization are poorly understood although locally producedfactors such as interleukin (IL)11 and activin (A)A regulate decidualization.We hypothesised that IL11 and AA interact to progress decidualization.Treatment with 0.5mM cAMP or 10-8mol/l estradiol 17b plus 10-7mol/lprogesterone (P) for 13 days, decidualized HESC. Decidualizing HESC weretreated with IL11 (100ng/ml), AA (50ng/ml), IL11+AA or control and mediacollected every 2 days to measure prolactin (PRL; a decidual marker), IL11and AA secretion. Non-decidualized HESC were treated with IL11, AA, or AAplus inhibitor (follistatin [200ng/ml], or SB542431, a TGFb1 receptor [Alk-5] inhibitor [10mM]) or control and IL11/AA secretion or phosphorylation(p) of the AA signalling component, SMAD2, measured.cAMP-induced HESC decidualization was biphasic, peaking on day (D) 8and D13 whereas P-induced decidualization was linear, peaking on D13.cAMP enhanced AA and P reduced IL11 secretion. Treatment with IL11 orAA significantly enhanced P-induced decidualization on D10 (AA) and D13(IL11 and AA; P<0.001). IL11+AA significantly promoted P-induceddecidualization above IL11 or AA treatment alone on D7 and D10 (P<0.05).AA induced pSMAD2 (P<0.005) and IL11 secretion (8.2�5.1 vs 1.9�0.7pg/106cells; P<0.05), which were blocked by inclusion of the AA inhibitors(P<0.005).This study demonstrates that IL11 and AA synergized to promote P- but notcAMP-induced decidualization. This suggests that the two in vitrodecidualization stimuli regulate decidualization via different mechanismsand demonstrates the importance in choosing an appropriate deciduali-zation stimulus to study the decidualization process. AA increased IL11secretion via pSMAD2 in non-decidualized HESC, suggesting AA is an earlyinducer of HESC decidualization. This data demonstrates that two locallyproduced cytokines interact to regulate decidual formation, an essentialcomponent in the development of a functional placenta.

N3.ADENOSINE A2B INCREASES L-ARGININE TRANSPORT AND NITRICOXIDE SYNTHESIS IN HUMAN PLACENTAL MICROVASCULARENDOTHELIAL CELLS FROM PREECLAMPTIC PREGNANCIES

C. Escudero*1,2, L. Myatt3, P. Casanello,1 L. Sobrevia1. 1Pontificia UniversidadCatolica de Chile, Chile. 2Universidad del Bıo-Bıo, Chile. 3University ofTexas Health Science Center, USA

This study aimed to characterize the role of adenosine receptor A2A and A2B

on L-arginine and nitric oxide (NO) synthesis in human placental micro-vascular endothelial cells (hPMEC).Methods: hPMEC from normal and preeclamptic pregnancies were iso-lated using positive immunoselection with CD31-magnetics beads. Kineticparameters for L-arginine transport, mRNA for human cationic amino acidtransporter 1 (hCAT1) and 2B (hCAT2B), activity and expression of induc-ible NO synthase (iNOS), nitrite and nitrotyrosine formation were deter-mined. Adenosine receptor involvement on L-arginine transport and NOSactivity was analyzed using agonists (CGS-21680 for A2A, NECA generaladenosine receptors) and the antagonists (ZM-241385 for A2A and A2B,MRS-1754 for A2B).Results: Preeclamptic derived hPMEC exhibits higher NOS activity (w4-fold), nitrite formation (w6-fold) and nitrotyrosine formation (w2-fold),associated with higher maximal velocity (Vmax) for L-arginine transport(w2-fold) but lower iNOS protein abundance (w50%), mRNA level (w40%)and iNOS promoter activity (w50%) compared with cells from normalpregnancies. In addition, preeclampsia was associated with higher hCAT2BmRNA level (w3-fold), without significant changes in hCAT1 mRNA. L-Lysine trans-stimulation of L-arginine transport was found in cells fromnormal pregnancies (w4-fold), but no significant alterations were detectedin preeclampsia. CGS-21680 restored L-arginine transport and NOS activityin preeclampsia to values in normal pregnancies. NECA did not alter L-arginine transport or NOS activity in preeclampsia, however increased(w2-fold) these parameters in cells from normal pregnancies. MRS-1754reduced L-arginine transport (w50 and 30%) and NOS activity (w70 and50%) in cells from preeclamptic and normal pregnancies, respectively. ZM-241385 reduced NOS activity (w75 and 55 %), but did not alter L-argininetransport in cells from normal and preeclamptic pregnancies, respectively.Conclusion: Stimulation of adenosine A2B receptor, rather than A2A, couldbe responsible for elevated NO synthesis and L-arginine transport viahCAT-2B in hPMEC from preeclampsia. This phenomenon could bea mechanism attempting to re-establish to normal values the reducedplacental vascular blood flow characteristic of preeclampsia.Supported by FONDECYT 1070865/1080534, CONICYT 24071039. C. Escu-dero holds PhD-MECESUP and Pontificia Universidad Catolica de Chilefellowships.


N4.FATTY ACIDS UPREGULATE THEIR OWN STORAGE INTO LIPID DROPLETSAND PROMOTE CELL AGGREGATION AND CYTOKINE PRODUCTIONINDEPENDENT OF GLUCOSE IN HUMAN CYTOTROPHOBLASTS

A.N. Pathmaperuma*1, P. Mana1, S.N. Cheung1, K. Kugathas1, M.E. Koina2,V. Delghingaro-Augusto4, D.A. Ellwood3, J.E. Dahlstrom2, and C.J. Nolan1,1Diabetes and Endocrinology Research Unit, 2Anatomical Pathology and3Fetal Medicine Unit, Australian National University Medical School at TheCanberra Hospital, Australia. 4Montreal Diabetes Research Center,University of Montreal, Canada

Aims: The diabetic pregnancy is characterized by maternal hyper-glycaemia and dyslipidaemia, such that placental trophoblast cells areexposed to both. The objective was to determine the effects of hyper-glycaemia, elevated non-esterified fatty acids (NEFA) and their interactionon trophoblast cell metabolism and function.Methods: Trophoblasts were isolated from normal term human placentasand established in culture for 16 h prior to experiments. Glucose utiliza-tion, fatty acid oxidation and fatty acid esterification were determinedusing radiolabelled metabolic tracer methodology at various glucose andNEFA concentrations. Lipid droplet formation, cell aggregation, viability,proliferation and apoptosis and the secretion of hormones and pro-inflammatory cytokines were also assessed.Results: Glucose utilization via glycolysis was near maximal at the lowphysiological glucose concentration of 4 mM; whereas NEFA esterificationinto triacylglycerol (TG) and diacylglycerol increased linearly withincreasing NEFA concentrations without evidence of plateau. Hyper-glycaemia caused intracellular glycogen accumulation, but had no othereffects on trophoblast metabolism or function. Culture of trophoblasts in0.25 mM NEFA for 24 h, however, upregulated fatty acid esterificationprocesses, inhibited fatty acid oxidation, inhibited glycerol release (amarker of lipolysis) and promoted lipid droplet formation, all consistentwith upregulation of fatty acid storage and buffering capacity. NEFA alsopromoted trophoblast aggregation and TNFa, IL-1b, IL-6 and IL-10production without effect on cell viability, proliferation, apoptosis orhormone secretion.Conclusion: NEFA have effects on trophoblast metabolism and function,independent of glucose, that may have protective as well as pathophysi-ological roles in diabetic pregnancy.

N5.SHED PLACENTAL ANTIGENS ARE CROSS-PRESENTED BY MATERNALDENDRITIC CELLS IN PREGNANCY

L. Moldenhauer*1, D. Thring2, J. Hayball2,3 and S. Robertson1. 1ResearchCentre for Reproductive Health, University of Adelaide, Australia; 2HansonInstitute, Australia; 3Sansom Institute, University of South Australia,Australia

The events through which the maternal T cell repertoire establishestolerance to paternally-derived antigens expressed by the placenta isunclear. To investigate this, we utilised a transgenic Act-mOVA malemouse expressing ovalbumin (OVA) ubiquitously as a model paternalantigen. OVA is inherited by the conceptus and expressed by placentaltissue. OVA-specific CD8+ OT-I T cells were adoptively transferred to Act-mOVA mated B6 females during pregnancy or post-partum. Extensive Tcell activation and proliferation was evident in all lymphoid tissue and thespleen from day 7.5 pc until 20 days post-partum, indicating systemicpresentation of placental OVA antigen. No activation of OT-I T cells wasdetected when bm1 mice, carrying a mutation in MHC class I H-2Kb, weremated with Act-mOVA males. This indicates that OVA-antigen presenta-tion depends upon processing by maternal antigen-presenting cells andcannot be presented by placental cells. Similarly, female mice witha mutation in the gene encoding transporter associated with antigenprocessing (TAP) failed to present placental OVA. When in vitro activated,cytotoxic OT-I T cells were adoptively transferred to pregnant femalescarrying OVA+ foetuses, fatal death occurred. This indicates that placentalcells express MHC class I–OVA peptide complexes that can be targeted bycytotoxic OT-I T cells. We conclude that placental antigens are processedand presented by professional, bone marrow-derived antigen presentingcells of maternal origin, presumably dendritic cells, via a TAP-dependentMHC class I pathway. While placental MHC class I-OVA complexes areexpressed by placental cells and can be targeted in T cell-mediated cyto-toxicity, placental cells do not present OVA antigen to elicit T cell activa-tion, likely due to the absence of placental cell expression of appropriateco-stimulatory molecules.


N6.THE EFFECT OF MALARIA INFECTION ON TERM PLACENTAL 11 BETAHSD2 EXPRESSION

Alexandra Umbers*, Caroline Clapham, Stephen Rogerson. University ofMelbourne, Adelaide

Introduction: Malaria is responsible for almost 1 million deaths per year,with pregnant women being particularly at risk of infection. Primigravidmothers are at greater risk of infection and severe disease and haveelevated serum cortisol levels compared to multigravidae. The fetus is atrisk of adverse birth outcomes including intrauterine growth restriction,low birth weight (LBW), preterm delivery, still birth and neonatal malaria.During infection Plasmodium falciparum infected erythrocytes concentratein the intervillous space and are often accompanied with inflammatorycells and elevated pro-inflammatory cytokines, both of which are stronglyassociated with LBW. The biological mechanisms that lead to adverse fetaloutcomes are not known, but placental insufficiency may underlie thepathogenesis. Placental 11-b hydroxy steroid dehydrogenase 2 (11-bHSD2)converts maternal cortisol to inactive cortisone, limiting transplacentaltransfer and fetal exposure to the growth restricting properties of cortisol.Because there is a well recognized relationship between elevated maternalcortisol and inflammation on 11bHSD2 function (and consequently birthweight) in other disorders of pregnancy, we assessed the relationshipbetween malaria infection and 11bHSD2 mRNA and protein levels.Method: mRNA and protein were measured using real time PCR andwestern blotting in placental biopsies from a cohort of 90 primigravidMalawian women with and without malaria infection.Results: Mean 11bHSD2 mRNA levels were reduced 40% compared touninfected controls. Moreover, 11bHSD2 mRNA was reduced 25% in LBWcases and 44% in mothers with more severe disease but differences did notreach significance. Protein analysis on 46 samples revealed a significantdecrease (68%) (p¼ 0.001) compared to controls. Furthermore, expressionwas reduced 54% (p¼0.15) in both LBW, and in 55% (p¼0.06) of motherswith severe disease.Discussion: This study reveals a significant reduction in 11bHSD2 andhighlights the potential role for excess fetal glucocorticoid exposure in thepathogenesis of fetal growth restriction observed in malaria duringpregnancy.

New Investigator Oral Session 2

N7.EFFECTS OF MATERNAL DEXAMETHASONE TREATMENT ON AMINOACID CLEARANCE AND DIFFUSIONAL EXCHANGE CAPACITY OF THEMOUSE PLACENTA DEPEND ON GESTATIONAL AGE

OR Vaughan*, AN Sferruzzi-Perri, PM Coan, GJ Burton & AL Fowden. Centrefor Trophoblast Research, Department of Physiology, Development andNeuroscience, University of Cambridge, UK

Introduction: Synthetic glucocorticoids, like dexamethasone (dex), aregiven to pregnant women threatened with pre-term delivery to improveneonatal viability. In experimental animals, this treatment restricts feto-placental growth but often increases fetal:placental weight ratio1.However, the effects of this treatment on placental phenotype remainunknown. Hence, this study examined the morphology and amino acidtransfer capacity of the mouse placenta after maternal dex administrationin the last trimester.Methods: Ad libitum fed pregnant C57/BL/6J mice were injected with dex(200ng/g, s.c., n¼24) or saline (n¼23) for 5 days before measurement ofunidirectional materno-fetal clearance of 14C-methyl-aminoisobutyric acid(MeAIB) under terminal anaesthesia at day (D) 16 (n¼18) or 19 of preg-nancy (n¼29; term 21days)2. Placental labyrinthine morphology wasassessed by stereology3. Differences between treatments were determinedby linear mixed model repeated measures ANOVA with Bonferroni post hoctest and considered significant when P<0.05.Results: On D16, placental but not fetal weight was significantly less in dexthan saline treated mice (Table 1). The fetal:placental weight ratio wasgreater in dex (4.4 � 0.1) than saline treated animals (3.9 � 0.1) (P<0.001).Fetal capillary volume, interhaemal membrane (IM) theoretical diffusioncapacity and MeAIB clearance were significantly higher in dex than salinetreated placentae at D16 (Table 1). On D19, both placental and fetal weightswere lower in dex than saline treated animals. Labyrinthine growth wasproportionally restricted in dex animals (Table 1). Neither labyrinthinearchitecture nor MeAIB clearance differed with treatment at D19 (Table 1).

Table 1 Fetal and placental weights (n¼8-15), placental morphology(n¼4-5) and MeAIB clearance (n¼7-12) of dex and saline treated mice atD16 and D19. *, P<0.05 vs control

D16Saline

D16 Dex
D19 Saline D19 Dex
Fetal Weight (mg)
394 � 5 398 � 6 1141 � 11 1106 � 11* Placental Weight (mg) 101 � 1 91 � 1* 86 � 1 78 � 1* Component Volume (mm3) Labyrinthine zone 43 � 2 41 � 1 48 � 1 44 � 1* Maternal blood space 4.6 � 0.5 5.1 � 0.1 7.5 � 1.5 8.7 � 0.7 Fetal capillary 2.9 � 0.2 3.9 � 0.3* 7.1 � 1.4 6.2 � 0.5 Labyrinthine trophoblast 36 � 2 33 � 0.1 33 � 3 29 � 1 IM Mean Surface Area (cm2) 7.6 � 0.8 10.9 � 0.1* 14.9 � 1.9 15.6 � 1.1 IM Harmonic Mean
Thickness (mm)
5.5 � 0.5 4.5 � 0.1* 3.8 � 0.4 3.5 � 0.1
Theoretical Diffusion Capacity(mm2min-1.kPa)

2.4 � 0.2
4.2 � 0.1* 7.1 � 1.3 7.8 � 0.8
MeAIB Clearance (ml.min-1.g-1)
38.0 � 2.0 52.1 � 1.7* 143.4 � 7.1 139.1 � 6.3
Discussion: The effect of maternal dex treatment upon placental pheno-type depends on gestational age. The small dex treated placenta adapts tomaintain fetal growth by altering amino acid transfer and labyrinthinemorphology at mid but not late gestation.References1. Fowden, AL. et al (2008) J Neuroendocrinol 20: 439.2. Sibley, CP. et al (2004) PNAS 101: 8204.3. Coan, PM. et al (2004) Biol Reprod 70: 1806.


N8.GENOME-WIDE DNA METHYLATION ANALYSIS OF FIRST TRIMESTERHUMAN PLACENTA

Boris Novakovic*1,2, Katrina Bell1, Hong Kiat Ng1, Andrew Sharkey3, AshleyMoffet3, Ursula Manuelpillai4, Eva Dimitriadis5, Jeff Craig1,2, RichardSaffery1,2. 1Developmental Epigenetics, Murdoch Children’s ResearchInstitute, Australia, 2Department of Paediatrics, University of Melbourne,Australia, 3Department of Pathology, University of Cambridge, UnitedKingdom, 4Monash Institute of Medical Research, Monash University,Australia, 5Prince Henry’s Institute of Medical Research, Australia

Epigenetic modifications, such as DNA methylation, play a major role incontrolling gene expression in tissues, and as such are involved in manyaspects of differentiation, development and function. The human placentais a unique organ in that it only exists for 9 months and displays manysimilarities to human tumours, such as growth in a low oxygen environ-ment, cell migration, and invasion of surrounding tissue and escape fromimmune detection. Not surprisingly therefore, the placenta also displaysa unique epigenetic profile with many similarities to the DNA methylationprofile of cancers including low global DNA methylation levels andhypermethylation of specific tumour-suppressor gene promoter regions.Our group, as well as others, has previously shown placental-specificmethylation of tumour suppressor genes, Ras-signalling inhibitors, Wnt-signalling inhibitors and genes regulating Vitamin D homeostasis. Thesespecific methylation patterns are likely to contribute to the developmentand function of the placenta; however, they are only a small part of theoverall placental epigenome. In order to determine the extent of placenta-specific methylation, an Illumina Infinium Human Methylation27 Bead-Chip array was used to detect genome wide methylation levels in firsttrimester (8 and 12 week) human placental tissue. Gene methylationstatus was then compared to three somatic tissues, and genes were clas-sified into one of four categories: (i) methylated in all tissues, (ii) specifi-cally methylated in the placenta, (iii) hypomethylated in all tissues, (iv)hypomethylated specifically in the placenta. Class (ii) genes were furthervalidated using the Sequenom Epityper quantitative methylation platform.This confirmed placenta-specific promoter methylation of genes involvedin several pathways, including multiple transcription factors, regulation ofapoptosis, oxidative-stress response, and cancer progression. We believethat disruption of these methylation patterns may contribute to poorplacental development and pregnancy-associated diseases such aspreeclampsia and IUGR.

N9.ANTIOXIDANTS REDUCE IMPACT OF ACTIVIN A INDUCED OXIDATIVESTRESS IN HUVECs

Rebecca Lim, Stephen Mandang, Pavitra Delpachitra, Rutu Acharya,Sebastian Hobson, Euan Wallace, Department of Obstetrics and Gynae-cology, Monash University, Australia

Background: The precise links between disturbed placental function andmaternal syndrome preeclampsia (PE) remain unclear. Maternal serumlevels of activin A increase 10-fold in PE and are increased months beforeclinical onset of signs and symptoms. We previously reported placentaloxidative stress as the likely cause of increased circulating activin. Morerecently, we have been exploring its possible systemic effects.Aims: Determine the role of activin A in the induction of endothelialdysfunction, specifically exploring endothelial cell oxidative stress,mechanisms of oxidative stress induction and efficacy of targetedantioxidants.Methods: HUVECs were cultured in the presence of activin A (50ng/mL) orpreeclamptic serum (10%) prior to treatment with follistatin 288 (FS;600ng/mL), tempol, superoxide dismutase or NOX inhibitor apocynin.Oxidative stress was assessed by ROS production and lipid peroxidationproduct, 8-isoprostane. Changes in NADPH oxidases (NOX) gene expres-sion were studied as likely mechanisms of oxidative stress. Endothelialdysfunction was assessed by ZO-1 expression and endothelialpermeability.Results: Activin A induces lipid peroxidation determined by increased8-isoprostane levels in a dose-dependent manner (*p<0.05) negated byFS. Compared to normal pregnancy serum, PE-serum increased ROS and8-isoprostane levels mitigated by FS. Similar trends were observed whenHUVECs were cultured in media containing PE serum, or PE serum and FS.ROS production was reduced in Activin treated HUVECs following expo-sure to Tempol, SOD and Apocynin. Following 2hrs stimulation withActivin, gene expression of ZO-1 was significantly reduced by approxi-mately 50% (**p<0.01), ET-1 expression increased >5-fold (*p<0.01), Nox2and Nox4 expression increased ~4-fold (*p<0.05) and 1.6-fold (*p<0.05)respectively while Nox1 and 5 remained unchanged compared tountreated HUVECs. Effects were mitigated by addition of Tempol, SOD orApocynin and related to endothelial integrity as determined by trans-endothelial permeability and resistances assays.Conclusions: Activin A directly induces oxidative stress in HUVECs, mostlikely through NOX activation and induction, thereby inducing increasedpermeability. The reduction in bioavailability of activin through addition ofFS or reduction of ROS production through administration of antioxidantsor NOX inhibitors may offer therapeutic potential for the treatment of PE.


N10.THE REGULATORY ROLE OF AP2a IN HUMAN EXTRAVILLOUSTROPHOBLAST INVASION

K. Biadasiewicz*, S. Sonderegger, S. Haider, P. Haslinger, M. Knofler.Department of Obstetrics and Gynecology, University of Vienna, Austria

Background: The AP2 family of transcription factors consists of five iso-forms: a-d. They are involved in important developmental and biologicalprocesses such as differentiation, proliferation, migration and invasion. Inthe placenta, AP2a and AP2g are predominantly produced in villoustrophoblasts. In particular, AP2a was shown to regulate transcription of thechorionic gonadotrophin subunits a and b. In immunohistochemical andgene chip analyses, we previously noticed that AP2a is also present inextravillous trophoblasts (EVTs), suggesting that it could be critical fortrophoblast invasion.Objective: To investigate the role of AP2a in controlling gene expressionand invasion of extravillous trophoblast cell models.Methods: Stable knock-down of AP2a was performed in the cell lineSGHPL-5 after infection with a murine stem cell virus (MSCV) – basedvector harbouring shRNAmir against AP2a. As a control SGHPL-5 cell poolscontaining a non-targeting shRNAmir were established. Primary EVTswere isolated from first trimester placentae and cultivated on Matrigelcoated dishes. Knock-down in EVTs was done with AP2a siRNA oligos(96h). Western blot analyses and real-time PCR were used to investigatepotential AP2a target genes. Proliferation was determined by countingcumulative cell numbers (4days). Invasion assays (24h) were done usingMatrigel coated transwells. Both functional assays were performed in theabsence or presence of EGF (5ng/ml).Results: Western blot data indicate that the extent of AP2a gene silencingwas approximately 70% in both the SGHPL-5 cell pool and EVTs comparedto non-targeting controls. Western blot and real-time PCR analysesrevealed upregulation of TIMP3 but downregulation of MMP2, MMP9, PAI-1, PAI-2, TIMP1, TIMP2, as well as the active form of uPA in the stable AP2aknock-down pool. Interestingly, the uPA pro-enzyme accumulated inSGHPL-5 knock-down cells suggesting that AP2a may affect uPA process-ing enzyme(s). So far, downregulation of MMP2 was also noticed uponAP2a gene silencing in EVTs. EGF-dependent invasion of the SGHPL-5 AP2ashRNAmir cell pool was significantly decreased compared to controls,proliferation was largely unaffected.Conclusions: AP2a controls expression of proteases and inhibitorscritically involved in EVT differentiation. Transwell assays with AP2aknock-down cells suggest that the factor could be important for growthfactor-dependent trophoblast invasion.

N11.SEX-SPECIFIC CYTOKINE RESPONSES OF PLACENTAE FROMPREGNANCIES COMPLICATED BY ASTHMA FOLLOWING IN-VITROLIPOPOLYSACCHARIDE STIMULATION

N. Hodyl*1, N. Scott2, A. Osei-Kumah1, M. Stark1 & V. Clifton1. 1RobinsonInstitute, University of Adelaide, Australia; 2The University of Newcastle,Australia

Introduction: Asthma affects 12% of pregnant women in Australia andresults in reduced female fetal growth. We have previously identified sex-specific alterations in placental cortisol metabolism and cytokine mRNAexpression with maternal asthma. We propose that activation of pro-inflammatory pathways in the placenta is therefore dependent on fetalsex.Hypotheses: We hypothesise that potentiated levels of pro-inflammatorycytokines will be evident in placentae of females compared to malesfollowing lipopolysachharide (LPS) stimulation in-vitro, and be furtherincreased in the presence of asthma. Additionally, females will exhibitgreater cortisol inhibition of cytokine responses than males.Methods: Placentae were collected from asthmatic (n¼12) and non-asthmatic women (n¼10). Placental explants were stimulated with LPS(1ng/ml) in the presence and absence of cortisol. Supernatant wascollected at 2 and 24 hours, and cytokine concentrations (tumour necrosisfactor (TNF) a, IL (interleukin) 1b, IL6, IL8, IL10) measured by ELISA(Luminex).Results: In control placentae post LPS stimulation, equivalent cytokineswere observed in both sexes. In the presence of asthma, female placentaeproduced greater IL1 and IL10 than male placentae (p<0.05). Only in thepresence of a female, TNFa, IL-1b and IL6 were increased in the asthmagroup at 2 hours (p<0.05), yet decreased at 24 hours relative to controls(p<0.05). Cortisol inhibited placental responses equivalently in placentaefrom both sexes at 2 hours.Conclusions: Increased pro-inflammatory cytokine production inplacentae was demonstrated only in the presence of a female fetus andmaternal asthma. This may contribute to the reduction in growth that wehave observed in female fetuses in the presence of asthma. As cortisolinhibition of cytokine production was observed in both male and femaleplacentae, the differential inflammatory responses between the sexes maybe due to other regulatory pathways, and is the focus of our ongoing work.


N12.GENOMIC INVESTIGATION OF A CONSERVED PLACENTAL GENENETWORK

E.B. Chuong*, D. Leuenberger, G. Bejerano, J.C. Baker, Department ofGenetics, School of Medicine, Stanford University, USA

The placenta exhibits striking structural and physiological diversity acrossmammalian taxa, and this variation is reflected in the evolution of manyspecies-specific placental genes. These apparent differences challengeresearch on pregnancy because the mouse model is presumed insufficientto represent human placentation. However, a growing number of keygenes including cdx2, hand1, and gcm1 have been observed to be funda-mental for placentation in all mammals, and our lab recently reporteda significant enrichment of conserved genes involved early placentaldevelopment (Knox and Baker, 2008). Therefore, we hypothesize that alleutherian mammals share a core genetic network that is conserved andessential for placental development.To investigate the placental gene network, we are utilizing next-genera-tion sequencing to assay genome-wide functional data over a timecourseof mouse placental development. We have begun carrying out high-throughput transcript tag quantification (RNA-Seq) to generate a compre-hensive transcriptome of placental development, and in parallel we areperforming genome-wide chromatin immunoprecipitation experiments(ChIP-Seq) to assay chromatin-level changes. Together these data willprovide a comprehensive picture of how placental genes are regulated ata global level. We have also developed computational methods to predictthe specific cis-regulatory elements that define the placental genenetwork, where we focus particularly on elements conserved across alleutherians. These elements may have emerged during the evolution of theancestral placenta and remained conserved due to their importance inplacental development. To test the ability of these elements to conferplacental expression in vivo, we have established a lentiviral transfectionreporter assay that is specific to the trophoblast lineage in mouse. Eluci-dation of the mammalian core placental gene network at the genomic levelwill provide novel insight into placental development, enable morefocused translational research using animal placenta models, and shedlight on the evolution of mammalian live birth.

Trophoblast Research Award

TR1.IMMUNE REGULATION BY CD4+CD25BRIGHT REGULATORY T CELLS ATTHE FETAL-MATERNAL INTERFACE DURING HUMAN PREGNANCY

Tamara Tilburgs1,2,3, Sicco A. Scherjon2, Barbara J. van der Mast2, GeertHaasnoot1, Minke Versteeg-v.d.Voort-Maarschalk1, Dave L. Roelen1, andFrans H. J. Claas1. 1Department of Immunohematology and Blood Trans-fusion and 2Department of Obstetrics, Leiden University Medical Center,Netherlands, 3Current work address: Department of Molecular andCellular Biology, Harvard University, USA

During pregnancy maternal lymphocytes at the fetal-maternal interfaceplay a key role in the immune acceptance of the allogeneic fetus. Recently,CD4+CD25bright regulatory T cells have shown to be concentrated indecidual tissue where they are able to suppress fetus-specific and non-specific responses. HLA-C is the only polymorphic classical histocompati-bility antigen expressed by fetal trophoblasts at the fetal-maternalinterface. Thus far no evidence has been provided that decidual T cellsspecifically recognize and respond to fetal HLA antigens at the fetal-maternal interface. In this study, we show that pregnancies containing anHLA-C mismatched child induce an increased percentage of CD4+CD25dim

activated T cells in decidual tissue. In addition, HLA-C mismatched preg-nancies exhibit a decidual lymphocyte response to fetal cells and containfunctional CD4+CD25bright regulatory T cells in decidual tissue, whereasHLA-C matched pregnancies do not. This suggests that decidual T cellsspecifically recognize fetal HLA-C at the fetal-maternal interface but areprevented from initiating a destructive immune response in uncompli-cated pregnancies.


IFPA Award in Placentology

L2.THE ROLE OF APOPTOSIS IN THE TURNOVER OF VILLOUS TROPHOBLAST

Berthold Huppertz, Medical University of Graz, Austria

Only about 15 years ago apoptosis has been attributed to the developmentof the human placenta. Even today a few scientists still ignore the over-whelming load of data that unambiguously show that programmed celldeath plays an essential role in placental growth and differentiation,especially in the villous trophoblast.Focussing on the apoptosis cascade within the villous trophoblast mygroup has detected the link between differentiation of cytotrophoblastsand syncytiotrophoblast on one hand and the progress of apoptosis on theother hand. The progress of differentiation of the villous trophoblast can betraced by following specific activities of proteins that are normallydescribed to become activated during the apoptosis cascade. Proteasessuch as caspase 8 but not calpains are used to trigger specific differentia-tion processes, and thus their activity is tightly regulated in terms of spaceand time.Dysregulation of processes such as proliferation, differentiation andapoptosis in the villous trophoblast may end up in pregnancy pathologiessuch as intrauterine growth restriction, preeclampsia or even spontaneousabortion. Especially preeclampsia seems to be closely related to themaintenance of the villous trophoblast. Under normal conditions apoptoticcorpuscular fragments are released from the syncytiotrophoblast (calledsyncytial knots), which are engulfed in the maternal lungs. Duringpreeclampsia this release shifts towards a release of non-apoptoticsubcellular fragments (even necrotic or aponecrotic), which is supposed toinduce the systemic inflammatory response of the mother typical forpreeclampsia.Today there is an intense search for markers to facilitate the earlyprediction of preeclampsia already during the first trimester of pregnancy.First very promising candidates such as placental protein 13 (PP13) arenow being launched for a general testing of pregnant women. I hope thatthis is the first step towards the identification of the origin of preeclampsiaand thus the first step towards strategies to prevent this syndrome.

L3.COMPLICATED INTERACTIONS BETWEEN GENES AND THEENVIRONMENT IN PLACENTATION AND PREGNANCY OUTCOME

Claire T. Roberts. University of Adelaide, Australia

Fetal programming can often be attributed to sub-optimal, but potentiallymodifiable, maternal factors such as smoking and poor nutrition. Much ofthe research in this field points to factors that cause intrauterine growthrestriction (IUGR) and the long term consequences for offspring health. It isnot greatly appreciated, however, that other complications that may occurwith, or independently of, IUGR predispose offspring, and their mothers, topoor health. Elevated maternal BMI increases the risk for most pregnancycomplications but it is clear that defects in placental invasion and functionpredispose to many adverse pregnancy outcomes. Our new data show thatpaternal obesity is associated with IUGR, independent of maternal BMI. Wehave also identified polymorphisms in a number of genes that regulatehow the placenta invades the decidua and differentiates, and how themother adapts to pregnancy, that are associated with poor outcomes.Excitingly, many of these are polymorphisms in the paternal genome. Onemight reasonably expect that these would be found in imprinted genesexpressed only from the paternal allele. However, we have also foundseveral non-imprinted genes in which paternal genotype has a significantinfluence on pregnancy outcome both on maternal and infant diseasestates, but also on fetal and placental growth parameters. Furthermore,these genes interact with the maternal environment including diet andsmoking to profoundly affect maternal and infant health. Consequently wenow propose a complicated model of the control of optimal placental andfetal growth and pregnancy outcome that includes important geneticcontributions from both parents to placental genotype that regulateconceptus growth and function. Importantly, paternal genotype caninfluence placental gene expression and the myriad of placental hormonesand growth factors secreted into the maternal circulation that modulatematernal adaptation to pregnancy and, in susceptible women, theseinteract with maternal genotype, BMI and lifestyle to cause poor maternaland infant health.


[P01.01].HDL: A NOVEL FETAL SIGNAL REGULATING PLACENTAL FUNCTION

Manuela Augsten*1, Birgit Ebner3, Angela Chemelli4, Uwe Lang1, GernotDesoye1, Christian Wadsack1. 1Clinic of Obstetrics and Gynaecology,Medical University Graz, Austria, 2Institute of Medical and ChemicalLaboratory Diagnostics, Medical Univeristy Graz, Austria, 3Center ofMedical Research, Medical University Graz, Austria, 4Institute of Chemistry,University of Graz, Austria

Introduction: Endothelial cells lining placental vessels are exposed tolipoproteins in fetal blood which differs from maternal circulation. Thepredominant fetal lipoprotein is high density lipoprotein (HDL), whichcontains more apolipoprotein E (ApoE) than adult HDL. We hypothesizethat ApoE has specific functions in the placenta. The aim was to identifygenes regulated by ApoE-rich HDL in primary endothelial cells isolatedfrom placental arteries (ECA) by incubating them with fetal HDL orreconstituted HDL-particles, following microarray analyses.Methods: Fetal HDL was isolated from cord plasma by ultrazentrifugation.Reconstituted HDL-particles were prepared of L-a-phosphatidylcholine,cholesterol and ApoE. Both were characterized by lipid electrophoresis andlight scattering. ECA (n¼5) were incubated with HDL or reconstituted HDL-particles for 16h versus untreated. RNA was controlled on BioAnalyzer.cDNA was hybridised onto microarrays (ABI) representing 29,098 humangenes.Results: The discoidal Apo-E rich particles had a size of 12.5nm in contrastto fetal HDL (17.5nm).Among 11.224 genes in the ECA transcriptome, 73 genes were differentiallyexpressed (p<0.05) when cells were exposed to HDL compared tountreated cells. 16 genes had a fold change <0.5 and 5 genes a fold change>2. When ECA were treated with reconstituted HDL-particles 1380 (12%)genes were regulated (p<0.05) versus untreated cells. Thereof 157 geneswere downregulated <0.5 fold and 359 genes upregulated >2 fold.Genes regulated by fetal HDL were clustered in lipid metabolism andsteroid biosynthesis, whereas those regulated by reconstituted HDL-particles participate in amino acid metabolism and stress response.32 (0.3%) genes were commonly regulated in both treatment groups. Theirfunctional clusters predominantly altered related to cell signalling (upre-gulated) and developmental processes (downregulated).Discussion: Fetal HDL alters gene expression in ECA. Some of thesechanges are induced by ApoE. HDL was identified as a novel fetal signalthat may regulate placental functions.(supported by grants 10053 to GD, 10896 to UH and 11165 to CW, OENBJubilee Fund)Keywords: primary placental endothelial cells, fetal high density lipo-protein (HDL), reconstituted HDL particle, gene expression

[P01.02].LPA PROMOTES MIGRATION OF HUMAN EXTRAVILLOUS TROPHOBLASTCELL LINE, HTR-8/SVneo

T Kotani*, S Sumigama, E Yamamoto, H Hayakawa, Y Mano, F Kikkawa.Nagoya Graduate University School of Medicine, Japan

Introduction: Successful pregnancy depends on the appropriate inva-sion of extravillous trophoblast (EVT) into the maternal decidua. Lyso-phosphatidic acid (LPA) is lysophospholipid mediators of diverse cellularprocesses exerted through a group of G protein-coupled receptors. It hasbeen reported to be involved in cell proliferation, cell survival, celldifferentiation regulation of gap junctions, stimulation of the serumresponse element, induction of inward ion currents, cell morphologicalchanges. LPA is important for blastocyst implantation in mice and sheep.In humans, serum lysophospholipase D activity increases during preg-nancy and LPA are involved in angiogenesis of first trimester decidua.However, the role of LPA in EVT function remains unknown. Therefore,we investigated the expression of LPA receptors and function of LPA inEVT, using human extravillous trophoblast cell line, HTR-8/SVneo.Methods and Results: RT-PCR analysis reveals that mRNA of LPA1 and LPA3receptors were identified in HTR-8/SVneo and primary EVT cells. Immu-noreactive LPA1 and LPA3 receptors were localized to EVT cells in firsttrimester implantation sites. LPA significantly stimulated invasion of HTR-8/SVneo cells. Moreover, LPA induced the phosphorylation of p42/44 MAPKin HTR-8/SVneo cells, which play roles in the stimulation of EVT motility.Conclusions: Our results suggested that LPA might stimulate human EVTinvasion with phosphorylation of p42/44 MAPK directly in placentation.Keywords: LPA, EVT, invasion, p42/44 MAPK

[P01.03].PLACENTAL UPTAKE OF POSTPRANDIAL VITAMIN A

Lesley Wassef1, Elena Giordano1,2, Loredana Quadro*1. 1Rutgers University,United States, 2University of Cagliari, Italy

Introduction: The developing mammalian embryo obtains vitamin A (VA)from the maternal bloodstream. Two majors circulating VA forms are:retinol (ROH) secreted from the liver stores bound to retinol-binding protein(RBP), in the fast state; retinyl ester (RE) within chylomicron lipoproteins ofintestinal origin, in the fed state. Both forms are used by the embryo to meetits VA needs (1). Intestinal chylomicrons are secreted into the bloodstreamand hydrolyzed by lipoprotein lipase (LPL) into smaller chylomicronremnants-RE. Although the liver takes up the majority of them, 25% is takenup by extrahepatic tissues. LPL catalyzes the hydrolysis of chylomicron-REand the amount of postprandial RE taken up by extrahepatic tissues corre-lates with LPL activity. LPL is expressed in placenta (2). We investigatedwhether LPL facilitates the uptake of postprandial VA in this tissue.Methods and Results: We showed that whole body lipase inhibition byadministration of P-407, followed by gavage of 3H-ROH of pregnant wild-type females, reduces the amount of postprandial VA taken up by thedeveloping tissues. Mice lacking LPL and overexpressesing human LPL onlyin muscle (MCKhL/LPL-/-) still produce chylomicron remnants RE, but donot express neither mouse nor human LPL in placenta (3). MCKhL/LPL-/-mice were crossed with mice lacking RBP (RBP-/-), which cannot mobilizeefficiently their VA stores and rely on dietary VA to support normalembryogenesis. MCKhLPL/LPL-/-RBP-/- mice were maintained on differentregimens of dietary VA during pregnancy (VA-sufficient, -deficient and-excess diet) and sacrificed at 14.5 dpc. MCKhLPL/LPL-/-RBP-/- embryoswere morphologically similar to RBP-/- embryos bred under similarmaternal dietary regimens. Also, steady state levels of maternal serum andembryonic VA levels in the MCKhLPL/LPL-/-RBP-/- strain were similar tothose of the RBP-/- control strain.Discussion: To date, our results suggest that systemic, but not placental-specific inhibition of LPL activity, affects uptake of postprandial VA in thistissue.

References1. Quadro L., et al. Mol Aspect Med 24, 421-430 (2003).2. van Bennekum et al., J Lipid Res 40, 565-574 (1999).3. Weinstock, P. H., J Clin Invest 96, 2555-2568 (1995).

Keywords: vitamin A, retinyl ester, lipoprotein lipase


[P01.04].A NOVEL ROLE OF CERAMIDE, SPHINGOSINE AND SPHINGOSINE-1-PHOSPHATE IN TROPHOBLAST DIFFERENTIATION

A.T. Singh*, A Dharmarajan, J.A. Keelan. University of Western Australia,Australia

Introduction: Differentiation and fusion of placental trophoblaststhroughout pregnancy is a unique process, resulting in formation ofa multinucleated cell membrane (syncytium). Critical mechanismsunderlying and regulating this fusion process remain unclear. Sphingoli-pids such as ceramide and sphingosine-1-phosphate have extensivelybeen associated with regulation of multiple biological activities, includingapoptosis, differentiation and proliferation. However, their role introphoblast differentiation has not yet been defined. We have previouslydemonstrated a correlation between trophoblast differentiation andceramide levels in transporter studies. Given that sphingolipids are keyplayers in early and late stages of apoptosis, which is an essentialcomponent of trophoblast differentiation, we hypothesized that theymight regulate trophoblast differentiation.Methods: Cultured cytotrophoblasts, isolated from term human placentasby enzyme digestion, were allowed to differentiate over 7 days in culture.Cell fusion commenced around day 2 of culture and was complete by day 4,according to hCG secretion levels.Results: Significant changes in intracellular levels of ceramide andsphingosine during differentiation were detected by LC-MS/MS. Expres-sion of enzymes of sphingolipid biosynthesis/metabolism also alteredsignificantly during differentiation and correlated with levels of theirsubstrates/products. Exogenous administration of a sphingosine kinaseinhibitor (which regulates S1P levels) significantly suppressed trophoblastdifferentiation, whereas modulation of ceramide or sphingomyelin levelsupregulated it.Conclusion: These findings are consistent with the hypothesis thatsphingolipid metabolism is involved in trophoblast differentiation andsyncytialisation. Dysregulation of ceramide-sphingosine metabolism bycellular stress modulators might have adverse effects on trophoblastdifferentiation and thereby placental function.Keywords: trophoblast differentiation, ceramide, sphingosine, sphingo-sine-1-phosphate

[P02.02].THE IMPRINTED AMINO ACID TRANSPORTER SNAT4 CONTROLS FETALGROWTH BY REGULATING UPTAKE OF AMINO ACIDS REQUIRED FORPLACENTAL GROWTH

E Angiolini1,2, R Smith1, K Hoelle2, I Sandovici2, H-W Yung2, G Konfortova1.1The Babraham Institute, United Kingdom, 2The University of Cambridge,United Kingdom

Imprinted genes are major regulators of fetal growth by controlling theprovision of maternal resources to the fetus. Slc38a4, which encodes theSystem A member SNAT4, is an imprinted gene abundantly expressed inthe placenta. Here, we show that a deletion of the predominant imprintedSlc38a4 transcript in mice leads to placental and fetal growth restrictionfrom mid-gestation. SNAT4 is essential for placental cell proliferationduring the peak phase of placental expansion and for volume regulation ofcells with important endocrine functions during late gestation. Unex-pectedly, we found little evidence that SNAT4 mediates the transfer ofamino acids to the fetus through the placenta. We propose that SNAT4plays an essential role in fetal growth by regulating amino-acid supply tokey placental cell-types and is a major determinant in the allocation ofmaternal resources within the placenta.Keywords: Genomic Imprinting, System A amino-acid transporters,Placental growth, Fetal growth

[P02.03].HEME OXYGENASE-1 GENE EXPRESSION IN UMBILICAL CORD,PLACENTA OF PREECLAMPSIA

Jong yun Hwang*, Ji yeon Lee, Sung Hun Na, Hyang ah Lee, Dongheon Lee.School of Medicine, Kangwon National University, Republic of Korea

Background: Heme oxygenase (HO) is an important enzyme to degradefrom heme to carbon monoxide, biliverdin and ferritin. HO and its prod-ucts are important oxidants to prevent tissue damage from oxidativestress. HO has three isoform enzymes. Especially, HO-1 is an inducibleform of the enzyme that is induced by heme, cytokines, endotoxins,hyperthermia and hypoxia.HO and its products play significant role in placentation, placental angio-genensis and antioxidant protection from oxidative stress.Preeclampsia (PET) is a hypertensive disorder in pregnancy. It is hypoth-esized that the cause of PET is disturbance of spiral artery modification oftrophoblast in the early placentation.I hypothesized that the deficiency of HO and its catalytic products may beassociated with PET and then adequate HO and its products may preventthe PET.Objects: The aim of this study was to evaluate the difference of the HO-1gene expression on placenta and umbilical cord between PET and normalpregnancy.Methods: In this study, we designed Case-control study including sevenwomen with PET. The umbilical cord and placenta tissue samples wereobtained from PET patients and normal delivery. We quantified HO-1 geneexpression on tissue samples by RT-PCR, real time PCR,immnunohistochemistry.Results: In our study, we observed a significant down-regulation of HO-1gene expression in umbilical cord and placenta when compared to normalplacenta and umbilical cord.Especially, HO-1 gene expression in the placenta decreased lower than it inumbilical cord.Conclusion: The present study demonstrates that the down-regulation ofHO-1 gene expression in placenta and umbilical cord is associated withpreeclampsia.Source of funding:‘‘This work was supported by the Korea Research Foundation Grant fundedby the Government(MOEHRD, Basic Research Promotion Fund) (KRF-2008-331-E00163)’’Keywords: heme oxygenase-1, preeclampsia, placenta, umbilical cord


[P02.04].THE DIFFERENT GENE EXPRESSION OF HO-1 AND iNOS IN PLACENTA OFINTRAUTERINE GROWTH RESTRICTION

Jong yun Hwang*, Ji yeon Lee, Sung hun Na, Tae gu Ahn, Dong heon Lee,Young Myeong Kim. Kangwon National University, Republic of Korea

Background: Heme Oxygenase (HO) and nitric oxide synthase (NOS) havesimilarities in some aspects; antioxidant function, generating gaseousmolecule (carbon monoxide, nitric oxide), similar isoform.HO and NOS play significant role in placentation, placental angiogenensisand antioxidant protection from oxidative stress in normal pregnancy.Some researcher suggested that HO and NOS may be regulated by mutualisoform enzymes.However, the exact comprehension for the compensatory regulationbetween two enzymes was deficient.Intrauterine growth restriction (IUGR) is a growth failure of the fetus. IUGRwas related to an inadequacy in the supply of nutrients and oxygen frommother to the fetus through the placenta. The common cause of IUGR isa placental insufficiency to result in hypoxia in fetus.We hypothesized that the deficiencies of HO, HO-catalytic products, NOSand NOS-catalytic products may result in IUGR and there is a reciprocalcompensatory system in two systems.Objects: The aim of this study was to identify the gene expression of HO-1and iNOS in placenta of IUGR and to evaluate the potential reciprocalcompensatory regulation between two systems.Methods: In this study, we designed Case-control study including womenwith IUGR. The placenta tissue samples were obtained from IUGR patientsand normal pregnancy. We quantified gene expression of HO-1 and iNOSon placenta by RT-PCR, real time PCR.Results: In this study, it demonstrates that a significant down-regulationof HO-1 gene expression in placenta when compared to normal placenta.However, we observed a significant up-regulation of iNOS gene expressionin placenta when compared to normal placenta.Conclusion: The present study demonstrates that there are differentresponses to placental insufficiency in two enzyme systems; the down-regulation of HO-1 gene expression and up- regulation of NOS geneexpression. We suggest that HO-1 and iNOS system may be regulated bymutual gene expression between two enzymes.Keywords: IUGR, Heme oxygenase, nitric oxide synthase, placenta

[P02.05].MICRORNA-mRNA NETWORKS IN THE LATE GESTATION MOUSEPLACENTA

W Kong*. University of Adelaide, Australia

Placental functional development is characterised by dynamic, co-ordi-nated changes in expression of many regulatory and functional genes andproteins that drive invasion, differentiation and growth. These changesmay arise in part from altered expression of microRNA (miRNA) regulatorynetworks. MiRNAs are short, single-stranded, non-coding RNAs involved inthe post-transcriptional repression of gene expression. MiRNAs bind tocomplementary sites in the 3’UTR of target mRNAs to repress or silencetranslation. This study aims to identify potential miRNA-mRNA regulatorynetworks that may be involved in the function of the mature mouseplacenta.In the current study, microarrays were used to compare miRNA and mRNAgene expression in the labyrinth and junctional zone of the late gestation(day 18) mouse placenta (term w19 days).13 miRNAs and 601 mRNAs were significantly differentially expressedbetween the labyrinth and junctional zone, as selected using Welcht-statistic. The interactions of miRNAs and their putative target mRNAswere modeled as their dependencies with Bayesian networks [1]. Thismethod integrates the prior targeting knowledge and expression profilesof miRNAs and mRNAs into Bayesian networks. Putative targets of miRNAswere extracted from freely accessible target databases, miRBase [2], PicTar[3], and TargetScan [4] and assessed by Ingenuity Pathways Analysis.Functional pathways involving the members of Bayesian networks in thejunctional zone included, integrin signaling, regulation of actin-basedmotility, IL-8 signalling (involving genes rhoc, rhod, rhov) and androgenand estrogen metabolism (involving genes hsd17b7, hsd3b4). This couldcorrespond to migration of glycogen cells into the decidua that occurs inlate gestation [5]. Significantly represented pathways found in thelabyrinth zone, included amino acid degradation and metabolism, fattyacid metabolism and synthesis and degradation of ketone bodies(involving genes acat1, hsd17b4). This may reflect the extensive metab-olism of nutrients in the labyrinth zone that occurs in the matureplacenta.References1. Pearl, J., ed. Probabilistic reasoning in intelligent systems: networks of

plausible inference. 1988, Morgan Kaufmann Publishers Inc.2. Griffiths-Jones, S., et al., miRBase: microRNA sequences, targets and gene

nomenclature. Nucleic Acids Res, 2006. 34(Database issue): p. D140-4.3. Krek, A., et al., Combinatorial microRNA target predictions. Nat Genet,

2005. 37(5): p. 495-500.4. Lewis, B.P., Burge, C.B. and Bartel, D.P. Conserved seed pairing, often

flanked by adenosines, indicates that thousands of human genes aremicroRNA targets. Cell, 2005. 120(1): p. 15-20.

5. Coan, P.M., et al., Origin and characteristics of glycogen cells in the devel-oping murine placenta. Dev Dyn, 2006. 235(12): p. 3280-94.

Keywords: MicroRNA, mouse, placenta, networks


[P02.07].E2-EPF UBIQUITIN CARRIER PROTEIN (UCP) MEDIATES HIF-1 INDUCEDGENE EXPRESSION IN CHORIOCARCINOMA CELL

T Sasaki*, H Nishi, J Liang. Tokyo Medical University, Japan

Introduction: E2-EPF ubiquitin carrier protein (UCP) is a member of an E2family of enzymes that catalyzes the ligation of ubiquitin to proteins targetedfor destruction by the proteasome. UCP is overexpressed in common humancancers, suggesting its involvement in oncogenesis, and detected coincidentlywith Hypoxia Inducible Factor (HIF)-1alpha in human primary liver, colon andbreast tumors, and metastatic cholangiocarcinoma. UCP targets the vonHippel-Lindau tumor suppressor, pVHL, for ubiquitin-mediated proteolysis incells, thereby stabilizing HIF-1alpha. We focused on preeclamptic placentaand examined the relationship between UCP and HIF-1alpha expression.Methods: Total RNAs were isolated from 42 normal placentas and 13preeclamptic placentas. Real-time reverse transcription-polymerase chainreaction (RT-PCR) was performed to evaluate the UCP mRNA expressionlevel. We examined the effect of overexpression of UCP on HIF-1alpha inchoriocarcinoma cell lines by western blot and luciferase assays usingluciferase constructs containing the Hypoxia Responsive Element (HRE).Results: Real-time RT-PCR analysis revealed that UCP mRNA expressionwas significantly lower in the preeclamptic placentas, compared withnormal placentas. By western blot analysis, UCP overexpression enhancedthe endogenous HIF-1alpha protein expression. Overexpression of UCPalso activated the transcriptional activity of the promoter containing theHRE by luciferase assays. Discussion: We found that UCP expression wasdown-regulated in preeclamptic placentas and its overexpression inducedHIF-1alpha expression in choriocarcinoma cell lines. Our results suggestthat high HIF-1alpha expression in preeclamptic placentas is regulated byreal hypoxic condition, not by UCP expression.Keywords: hypoxia, transcription, preeclampsia

[P02.08].PLACENTAL LACTOGEN FUNCTION IN POST-IMPLANTATION MURINEPREGNANCY

S M Rawn*, J C Cross. University of Calgary, Canada

Introduction: In rodents, the Placental Lactogen (PL) hormones areproduced by the placenta, detectable in the blood of pregnant mothersfrom mid-gestation and act through the Prl receptor (PrlR). PL knockoutmice have not been reported, but is complicated because there are four PLgenes. Prl and PrlR mutant female mice are infertile but in PrlR mutants(129SvJ genetic background) their implantation defect can be rescued byexogenous progesterone. Our aim was to infer PL function by comparingthe phenotypes of Prl-/- and PrlR-/- mice, as any differences must be due toeffects of PLs.Methods: Maternal parameters (blood pressure, blood glucose, metabo-lites, spleen weight), implantation rates and fetal growth were observed inPrl-/- and PrlR-/- mice supplemented with progesterone. To account forpossible genetic background effects, both mutations were maintained inthe C57/Bl6 strain. All females were mated to wildtype C57/Bl6 males.Results: 1) In the C57/Bl6 background, whereas Prl-/- mice were able tocarry pregnancies to term, none of the PrlR-/- mice have been able toestablish a pregnancy to date.2) Pregnant Prl-/- females had normal blood pressure and blood glucose.3) In Prl-/- mice, w50% of fetuses detectable at mid-gestation survived to term.4) All fetuses in Prl-/- mothers had reduced heart rates and crown-rumplengths at mid-gestation and were leaner at term.Discussion: Our major conclusion is that maternal Prl is not required forpregnancy if implantation is rescued with progesterone. The greaterseverity of the PrlR mutant phenotype implies that PLs, acting through thePrlR, are essential for establishment and/or maintenance of pregnancy. Ofinterest, embryos in Prl-/- females are developmentally delayed. Whetherthis is related to Prl deficiency or progesterone supplementation is unclear.Unexpectedly, genetic background affects the PrlR mutant phenotype suchthat it is less severe in 129SvJ compared to the C57/Bl6 strain.Keywords: Placental Lactogen, Prolactin, post-implantation, develop-mental delay

[P02.09].GLUCOCORTICOIDS REPRESS INDUCTION OF CYP1A1 GENEMEDIATED BY ARYLHYDROCARBON RECEPTOR (AhR) LIGANDSMETHYLCHOLANTHRENE (MC) OR 2,3,7,8-TETRACHLOROBENZO-P-DIOXIN (TCDD) IN PLACENTAL JEG-3 CELL LINE

Lucie Stejskalova*1, Katerina Pospechova1, Lucie Svecova1, Radim Vrzal2,Zdenek Dvorak2, Petr Pavek1. 1Department of Pharmacology and Toxi-cology, Faculty of Pharmacy in Hradec Kralove, Charles University in Pra-gue, Czech Republic, 2Department of Cell Biology and Genetics, Faculty ofSciences, Palacky University in Olomouc, Czech Republic

CYP1A1, an enzyme of the cytochrome P450 family, is the most importantbiotransformation enzyme in the placental barrier for which relevantinducible activity has been demonstrated in the placental trophoblastthroughout pregnancy. CYP1A1 metabolizes several drugs widely used inpharmacotherapy. At the same time CYP1A1 plays a key role in bio-activation of procarcinogens and proteratogens such as polycyclic aromatichydrocarbons (PAHs) to form DNA-adducts, which bind to placental andfetal DNA and damage it. Expression of CYP1A1 is transcriptionally regu-lated through the ligand-activated aryl hydrocarbon receptor (AhR). It hasbeen reported that glucocorticoids (synthetic and endogenous) modulatethe induction of CYP1A1 via activated AhR.The aim of this work was to study the cross-talk between arylhydrocarbonreceptor and glucocorticoid receptor in transcriptional regulation ofCYP1A1 in placental JEG3 cell line exposed to AhR ligands methylcholan-threne (MC) or 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) alone and incombination with glucocorticoid dexamethasone.The effect of dexamethasone on ligands-mediated transactivation ofCYP1A1 was assessed employing real-time RT-PCR and gene reporter assayin transiently transfected cells with luciferase gene reporter plasmidscontaining native promoter or responsive sequences of CYP1A1 gene. Inaddition, nuclear translocation of AhR/ARNT was monitored using Westernblotting.Our preliminary results show that 24-h treatment with dexamethasonedecrease AhR-mediated transactiovation of CYP1A1 gene in gene reporterassay with both p1A1-luc and pXRE-luc reporter plasmids and that thearomatic hydrocarbon-induced CYP1A1 mRNA was suppressed by dexa-methasone. We show that the phenomenon is likely due to decreasednuclear translocation of AhR after treatment with dexamenthasone. Basedon our preliminary data, we can suggest cross-talk of GR/AhR pathways ingene regulation of CYP1A1 in placental trophoblast, which is not likely atthe level of transcriptional regulation.AcknowledgementThis project was supported by the grant number GAUK 170/50/95003.Keywords: arylhydrocarbon receptor, glucocorticoid receptor, cytochromeCYP1A1, TCDD


[P02.10].MICRORNA hsa-miR-517a IS POSSIBLY INVOLVED IN TUMOR NECROSISFACTOR (TNF)-MEDIATED SIGNALING IN HUMAN PLACENTA

T Takizawa*, O Ishibashi, T Ishikawa, G Ishikawa, A Katayama, T Takeshita.Nippon Medical School, Japan

Objectives: MicroRNAs (miRNAs), small noncoding RNAs, are involved ina variety of biological processes in animals. However, little is known aboutthe role of miRNAs in physiological and pathological processes duringpregnancy. Recently, we found that human placental trophoblast cellsexpressed placenta-specific miRNAs (e.g., hsa-miR-517a). In this study, weperformed a proteome analysis by miRNA overexpression, usinga trophoblast cell line, to elucidate the biological function(s) of placenta-specific miRNAs.Methods: BeWo cells were transfected with Pre-miR hsa-miR-517a miRNAPrecursor Molecules (Applied Biosystems). The transfected cells wereharvested 24-72 h after transfection, and subjected to proteome analysis.Cell lysates were subjected to two-dimensional difference gel electro-phoresis (2D-DIGE). Proteins in the spots were analyzed using a LC/MSmass spectrometer. Data from mass spectrometry were analyzed usingMASCOT software, and proteins hit with significantly high scores weresubjected to IPA (Ingenuity Systems) to create possible networks/pathwaysinvolving the proteins.Results: Our proteome analysis by 2D-DIGE resulted in a total of 40 spotswith differential intensities between BeWo cells transfected with hsa-miR-517a and the negative controls. Subsequent mass spectrometry analysisidentified a total of 58 proteins as promising candidates of hsa-miR-517a-regulated proteins. Although none of these proteins are included in the insilico targets of hsa-miR-517a, a network-based analysis using IPA sug-gested that most of the hsa-miR-517a-regulated proteins were mapped totwo networks. The first network is NFKB1- and MAPK-related signaling;the second network TNF-related signaling. Since TNF elicits the activationof both MAPK and NFKB1accompanied by apoptosis induction in manytypes of cells, the two networks can be merged.Conclusions: A possible function of hsa-miR-517a could be to regulatesignal transduction mediated by TNF and/or other death ligands, althoughmore detailed studies are necessary. Our data provide new insights intomiRNA biology of the human placenta.Keywords: human placenta, trophoblast, microRNA, TNF

[P02.11].MICRORNA EXPRESSION CHANGES DETECTED BY MICROARRAY WITHFORSKOLIN SYNCYTIALISATION OF BeWo CELLS

Y Gao1,3, UW Nilsson1,2, C Whitehead1,2, S Tong*1,2. 1Centre for Women’sHealth Research, Monash Institute of Medical Research, Australia, 2Centrefor Cancer Research, Monash Institute of Medical Research, Australia, 32ndAffiliated Hospital, Xian Jiaotong University School of Medicine, China

Introduction: Syncytialisation is a process of cytotrophoblast maturationwhere cells undergo cytoplasmic fusion. The resulting syncytium isa multinuclear structure lining the fetoplacental interface, and playscrucial endocrine, immune quenching and nutrient exchange roles.MicroRNAs (miRNAs) are endogenously produced and non-coding RNAsthat bind to the 3’-untranslated region of mRNAs, suppressing translation.They represent a new level of genetic control, possibly regulating a third ofall genes. miRNAs highly expressed in the placenta, and putative placentalspecific miRNAs have been reported1,2.We set out to characterise changes in miRNA expression withsyncytialisation.Methods: We used cultured BeWo cells (placental derived cells fromchoriocarcinoma), syncytialised in 100uM of forskolin and left in 37�C for48 hours (n¼4 per condition). Syncytialisation was confirmed by increasedhCG levels in the supernatant (51,441�1,551 IU/L with forskolin, vs 3,919�246 IU/L for controls) and loss of E-Cadherin staining on immunohisto-chemistry (Figure 1).Total RNA was extracted using Trizol reagent. RNA purity and quality wasconfirmed with the Experion system (Bio-Rad). A miRNA array (Exiqon)was performed on all samples, with >500 human miRNAs represented.

Results: Of 1,537 miRNAs on the array, there were 18 human specificmiRNAs that showed significantly different changes in expression (Table 1).Eleven were downregulated. Of these, six were previously shown to behighly expressed in the placenta with three [miR 20, 492, 584] beingpossibly placental specific (4-6 fold higher than all other tissuesscreened1,2). Of seven miRNAs that were upregulated, four were previouslyshown to be expressed in placenta with one being possibly placentalspecific [miR 23a]1,2.Conclusion: Syncytialisation is associated with rapid changes of placentalexpressed miRNAs, including miRNAs possibly unique to placenta. miRNAsmay play crucial roles in syncytialisation.References1. Bentwich et al. Nature Genetics 2005;37(7):766-770.2. Barad et al. Genome Research 2004;14:2486:2494.

Table 1: miRNAs that changed in expression with syncytialisation.

miRNAs not miRNAs reported to miRNAs that may be placental
specific toplacenta
bi

e highly expressedn placenta

sp
pecific (ref 1,2) – subset ofrevious column
Downregulated
miR–32 m iR–20a m iR-20a miR–175b m iR–20b m iR-492 miR–422b m iR–21 m iR–584 miR–565 m iR–32 miR–654 m iR–183
m
iR–492
Upregulated
miR–455 m iR–23a m iR-23a miR–483 m iR–23b miR–637 m iR–326
m
iR–513
Keywords: microRNA, syncytialisation, BeWo cells, microarray


[P02.12].THE IMPRINTED GENE Phlda2 REGULATES THE GLYCOGEN CELLPOPULATION OF THE MOUSE PLACENTA

S J Tunster*1, B Tycko2, R M John1. 1Cardiff University, United Kingdom,2Columbia University, United States

Introduction: Genomic imprinting is proposed to account for the transi-tion from a relatively primitive non-invasive placenta in marsupials to themore complex and invasive placenta characteristic of eutherian mammals(John and Surani 2000; Kaneko-Ishino et al. 2003). The maternallyexpressed imprinted gene Phlda2 has previously been shown to regulateplacental development, with loss of expression associated with placento-megaly due to a disproportionate expansion of the spongiotrophoblastlayer (Frank et al. 2002). Here we further characterise the effect of over-expressing this gene in the mouse placenta.Methods: We have previously briefly reported on transgenic mice thatcarry 3 copies of a BAC transgene spanning the imprinted gene Phlda2(Salas et al. 2004). Here we present the detailed characterisation ofplacental development in response to incrementally increasing dosage ofPhlda2 on both 129/Sv and mixed 129/Sv x C57BL/6 genetic backgrounds.Results: We directly show that placental stunting in Phlda2 transgenicmice is due to a disproportionate loss of the junctional zone. A defect in theectoplacental cone is apparent from E10.5 with a loss of the proliferatingcomponent. Late in development there is a striking reduction in glycogencell staining and at E16.5 the glycogen cells fail to migrate to the maternaldecidua in a timely manner. Instead, Tpbpa-positive cells progressivelymislocalise in the labyrinth layer. Furthermore, embryonic growth isadversely affected from E16.5 with transgenic animals born w13% lighterthan wild type.Conclusions: Glycogen cells may represent a source of readily metabolisedenergy during the final stages of gestation when the embryo is placing thehighest demand on maternal resources (Coan et al. 2006). Over-expressionof Phlda2 is associated with a reduced glycogen cell population. Imprintingof Phlda2 may be responsible for the expansion of this cell type thuspermitting prolonged gestation and the birth of developmentally matureprogeny.Keywords: Imprinting, Glycogen cells, Placenta, IUGR

[P02.13].BIPARENTAL EXPRESSION OF HUMAN CGB GENES IS REQUIRED FORSUCCESSFUL IMPLANTATION

L Uuskula*1, K Rull1,2, L Nagirnaja1, M Laan1. 1Institute of Molecular and CellBiology, University of Tartu, Estonia, 2Department of Obstetrics andGynecology, University of Tartu, Estonia

Introduction: Placentally expressed Chorionic Gonadotropin Hormone(hCG), composed of a and b subunits, has an irreplaceable role in theimplantation of an embryo in primates. In human, the four genes codingfor hCG and luteinizing hormone beta subunits are co-located in the LHB/CGB gene cluster at 19q13.32. Several genes expressed in placenta andessential for reproduction are known to be imprinted. This study aimed to(i) explore the parental origin of hCG beta transcripts; and (ii) compareDNA methylation status of hCG beta promoters between trophoblastictissue and blood leukocytes. Chorionic Gonadotropin beta 5 (CGB5) andChorionic Gonadotropin beta 8 (CGB8) genes were used as a model sincethese two loci are contributing 2/3 up to 3/4 of total hCG beta mRNAtranscripts.Study groups: We analysed trophoblastic material from 23 mother-offspring duos and 9 mother-father-offspring trios including cases of (1) IIItrimester normal delivery at term (n¼14), (2) I trimester elective termi-nation of pregnancy (n¼10) and (3) recurrent (�3) miscarriages (RM)during I trimester of pregnancy (n¼8).Methods: (1) Parental origin of the transcribed alleles of CGB5 and CGB8was determined using gene-specific RT-PCR, cloning and sequencing. (2)Methylation status of a CGB5 promoter CpG island was assessed bymethylation-specific PCR after bisulphite treatment of the genomic DNA,followed by cloning and sequencing. (3) Large chromosomal rearrange-ments were detected using Illumina CNV370-Duo array system.Results: (1) Normal successful pregnancy requires biparental expression ofthe CGB5 and CGB8 genes. (2) Trophoblastic material of some RM casesrevealed an allelic imbalance – only maternally inherited CGB5 gene alleleswere expressed. (3) RM cases with uniparental CGB5 gene expression werecharacterized by hemimethylated CGB5 promoter as well as abundantchromosomal arrangements across genome.We hypothesize that chromosomal abnormalities (e.g. aneuploidy) mayaffect epigenetic programming during embryonic development.Keywords: reproduction genetics, chorionic gonadotropin genes, DNAmethylation


[P02.14].DIFFERENTIALLY METHYLATED REGION IN INTRON 1 OF STOX1EXPLAINS PARENT-OF-ORIGIN EFFECT SEEN IN PRE-ECLAMPSIALINKAGE ANALYSIS

M van Dijk*, CBM Oudejans. VU University Medical Center, Netherlands

Introduction: The 10q22 chromosomal region with genomic linkage topre-eclampsia shows a parent-of-origin effect with maternal trans-mission1,2. By studying the methylation status of the CpG island within theSTOX1 promoter region, we and others3,4 have been unable to identifya differentially methylated region (DMR) explaining this parent-of-origineffect. Recently, in intron 2 of stox1 in mice, a tissue-specific DMR has beenidentified5. Furthermore, in human lymphoblastoid cells a differentialallelic expression ratio was detected for STOX1 SNPs6. Both of these studiesindicate the existence of a DMR in humans leading to differentialexpression, and potentially explain the parent-of-origin effect observed.Methods: By computational analysis a region within intron 1 of the STOX1gene was identified as a potential CpG island. This was confirmed using CT-converted DNA of buffycoat samples as a source of cells in which differ-ential expression was observed6. Normal and androgenetic early placentasamples were tested in the same manner along with term placenta,SGHPL5 cells and adult tissue samples.Results: The methylation pattern identified in term placenta and adulttissue is consistent with an imprinted gene in which 50% of the alleles aremethylated in tissues expressing STOX1, while 80% of the alleles aremethylated in tissue with very low levels of STOX1 expression (liver). Inearly placenta only 20% of the alleles were methylated, while androgeneticplacentas, consisting of paternal alleles only, showed 50% methylation.SGHPL5 cells, representing extravillous trophoblasts, were also 50%methylated. This indicates that in early placenta, in contrast to term andadult tissue, only some cell types (extravillous trophoblasts among others),are subject to imprinting.Conclusion: This study confirms that the parent-of-origin effect observedin the pre-eclampsia linkage analysis is caused by a DMR within STOX1,leading to cell type-specific imprinting in the early placenta.References1) Oudejans et al., Hum Mol Reprod 2004.2) van Dijk et al., Nat Genet 2005.3) van Dijk et al., Nat Genet 2007.4) Iglesias-Platas et al., Nat Genet 2007.5) Suzuki et al., Genes Cells 2007.6) Cheung et al., Am J Hum Genet 2008.Keywords: pre-eclampsia, STOX1, parent-of-origin, differentially methyl-ated region

[P02.16].FEATURES OF CONSTITUTIVE HETEROCHROMATIN REGIONSEXTRAEMBRYONIC AND EMBRYONIC TISSUE IN HUMAN EMBRYOS

Trofimova Irina*, Kuznetzova Tatyana. Saint-Petersburg State University,Russian Federation

Epigenetics modifications of chromatin are methylation DNA and histonemodification. Centromeric chromatin is constitutive heterochromatinregions (CHR). DNA located in this site of chromosome, belongs to the classof satellite DNA. The implications of epigenetic silencing in normaldevelopmental gene regulation, aging and cancer progression have madeheterochromatin the focus of intense investigation. Special interest isrepresented large CHR autosome 1, 9, 15,16, 22 and Y-chromosomes ofhuman in which presence of the structural genes is supposed and func-tional value is discussed.CHR in chromosomes of different tissues are characterized by tightcondensation, late replication and methylation. Meanwhile decondensation,hypomethylation CHR in human trophoblast and tumorcells were registered.Stress-induced transcription of satellite III in some cell lines was shown.We studied definite peculiarities of CHR in direct and semi-direct slidesfrom chorionic villi and embryonic tissue samples (8 and 3 accordingly)from artificial abortions after pretreatment of slides with differentenzymes (RNase A, DNase I, DNA- ligase T4) and dyeing by acridine orange.Separate and combined pre-treatment of slides with different nucleases(RNase A and DNase I) observed of red fluorescence of constitutiveheterochromatin (typical for single-stranded DNA and RNA) in 1qh, 9qh,16qh, Yqh, 15cenh, 22cenh most metaphase plates. Chromosome armswere relatively uniform stained or banded similar to RFA in some meta-phases. Subjection to DNA -ligase T4 resulted in total absence of fluores-cence ("black holes") in CHR and uniform green fluorescence alongchromosome arms. Staining pattern corresponded to untreated slides wasobserved in all control slides treated with corresponding buffer solutions.Our results advocate for unusual conformation packaging of CHR whichcould be attributed to the feasible transcriptional activity of pericentro-meric satellite DNA in embryonic and cytotrophoblast cells duringembryogenesis.


[P02.17].UNIQUE PROFILE OF DNA METHYLTRANSFERASE PROMOTERMETHYLATION IN HUMAN PLACENTAL TISSUE AND PURIFIED FIRSTTRIMESTER TROPHOBLASTS

B Novakovic1,2, N Wong1,2, M Sibson1, J Craig1,2, R Saffery*1,2. 1MurdochChildrens Research Institute, Australia, 2University of Melbourne, Australia

One of the earliest lineage differentiation events in embryogenesisinvolves the formation of extraembryonic and embryonic lineages in theblastocyst. This is accompanied by a dramatic change in global DNAmethylation levels, such that extraembryonic (and placental) tissue isglobally hypomethylated in relation to all somatic tissues. Despite this, it isbecoming increasingly clear that the placenta also shows a unique profileof gene specific hypermethylation.The family of DNA methyltransferases are responsible for both the main-tenance (DNMT1) and de novo establishment (DNMT3a, -3b, -3L) of DNAmethylation during cell division and development. In order to examine thepotential role of these genes in regulating the placental epigenome, weinvestigated the promoter methylation status of each in human placentaltissue and purified cell populations using state-of-the-art SequenomMassARRAY EpiTyping. No promoter methylation of DNMT3A or -3B wasseen in any tissue tested. Intriguingly however, both full term and firsttrimester placenta show specific hypermethylation of the maintenanceDNA methyltransferase-1 (DNMT1) gene and hypomethylation of theDNMT3L gene that modulates DNMT3A and -3B- mediated de novo meth-ylation. This was confirmed by bisulphite sequencing methylation analysiswhich also revealed monoallelic methylation of DNMT1 in villous tissueand purified cytotrophoblasts, with no evidence of imprinting (parent oforigin effect). DNMT1 was not methylated in any somatic tissue testedwhile DNMT3L is hypermethylated and silenced in most somatic tissues. Invitro methylation experiments confirmed that DNMT1 promoter methyl-ation attenuates transcriptional activity in human trophoblasts. Thispattern of concomitant epigenetic down-regulation of the maintenanceDNMT1 gene with up-regulation of the DNMT3L regulator of de novomethyltransferases is anticipated to have a profound effect on the estab-lishment of the placental epigenome.Keywords: DNA methylation, DNMT1, DNMT3L, Epigenome

[P03.01].ALTERED PLACENTAL GENE EXPRESSION IN RESPONSE TO MATERNALDEXAMETHASONE EXPOSURE IN THE MOUSE

JSM Cuffe*1, H Dickinson2, WM Boon2, KM Moritz1,2. 1School of BiomedicalSciences, The University of Queensland, Australia, 2Department of Physi-ology, Monash University, Australia

Introduction: Increased maternal glucocorticoid exposure has been shownto program adult onset disease in offspring. Previous studies have utilizeda number of animal models to look at the effects of short term maternalglucocorticoid exposure, but rarely has this been done in the mouse. Wetested the hypothesis that a short term maternal dexamethasone exposureduring pregnancy in the mouse would affect the function of the developingplacenta which may contribute to the programming of disease.Methods: C57/BL/6 mice were infused with dexamethasone (DEX) orsaline (SAL) for 72 hours via osmotic minipump beginning at embryonicday (E) 12.5. Placentas were collected at E14.5 (during infusion) or E17.5(after infusion) and embryo and placental weights recorded. Geneexpression of GLUT1, GLUT3, Map2k1 and 11bHSD2 was examined by realtime PCR.Results: Placental and foetal body weights tended to be reduced inresponse to DEX at E14.5 (P¼0.06), however at E17.5 there was no differ-ences between groups in body or placental weight. DEX caused significantalterations in gene expression as shown in the table below:

Relative gene expression: (n¼9-10 samples from 4-6 litters, *P¼<0.05)

Gene SAL DEX

GLUT1
E14.5 1.15�0.22 2.62�0.49* E17.5 1.02�0.07 1.54�0.49
GLUT3
E14.5 1.05�0.10 1.15�0.08 E17.5 1.02�0.06 0.72�0.07*
Map2k1
E14.5 1.10�0.15 1.73�0.24* E17.5 1.01�0.06 0.71�0.08*
11bHSD2
E14.5 0.99�0.47 2.50�0.44 E17.5 1.00�0.10 4.05�1.70
Conclusion: Using our novel mouse model of short term DEX exposure, weshow a transitory reduction in both placental and foetal weight indicatingDEX has directly inhibited growth and development. However, normalweight two days after completion of treatment suggests significant catchup growth. Changes in glucose transporter gene expression suggestalterations in placental and foetal nutrient supply while changes inMAP2k1 expression suggests altered placental vasculogenesis. Thetemporal changes in expression demonstrate direct and compensatoryeffects of DEX exposure.Keywords: Glucocorticoids, mouse, placenta, genes


[P03.02].ALTERED GLUCOCORTICOID METABOLISM IN PLACENTAL TISSUEFROM FIRST TRIMESTER PREGNANCIES AT INCREASED RISKOF PRE-ECLAMPSIA

S Mukherjee*, JE Cartwright, G Whitley StJ, AE Michael, B Thilaganathan.St George’s, University of London, United Kingdom

Introduction: Glucocorticoids may exert important actions in the placentain early pregnancy. The local actions of glucocorticoids can be modulatedby 11b-hydroxysteroid dehydrogenase (11bHSD) enzymes which catalyseinter-conversion of cortisol with its inert metabolite, cortisone. Although,11bHSD isoenzymes are known to be expressed in the placenta anddecidua, their expression and activity have not been well characterised inthe first trimester placenta. Measurement of uterine artery resistanceindices (RI) by Doppler Ultrasound in the first trimester can be used toassess the risk of developing pre-eclampsia. The aim of this study was tocompare 11bHSD expression and activity in first trimester placental tissuefrom pregnancies screened as being at the highest and lowest risk ofdeveloping pre-eclampsia.Methods: Expression of 11bHSD enzymes was assessed in first trimesterplacental tissue by western blot analysis and immunohistochemistry.Enzyme activities were assessed using radiometric conversion assays.Results: Western blot analysis confirmed expression of 11bHSD2 in firsttrimester placental tissue and immunohistochemistry localised expressionof the 11bHSD2 protein to the syncytiotrophoblast. 11bHSD1 expressionwas not detected in chorionic villous tissue. Enzyme activity assaysconfirmed NAD+-dependent inactivation of cortisol by 11bHSD2 in firsttrimester placental tissue. Net cortisol oxidation was significantly greaterin placental tissue from pregnancies at higher risk of pre-eclampsia than inlow risk pregnancies (50.9�15.9 versus 18.3�1.9 pmol cortisone/mgprotein, n¼11 & 12, respectively; p<0.05).Discussion: 11bHSD2 expression is thought to protect the fetus fromexposure to maternal cortisol. While other studies have suggested that11bHSD2 is downregulated in term pre-eclamptic placentae, our studiessuggest that there is increased activity in first trimester placenta frompregnancies at higher risk of developing pre-eclampsia. It remains to bedetermined if it is related to the pathopysiology of pre-eclampsia or isa compensatory response to poor trophoblast development.Keywords: Trophoblast, Glucocorticoids, Pre-eclampsia, Uterine Dopplers

[P03.03].CORTICOSTEROIDS UP-REGULATE HYPOXIC, APOPTOTIC ANDPLACENTAL GENES IN SEVERE GROWTH RESTRICTION: GENOME-WIDETRANSCRIPTIONAL PROFILING OF mRNA TRANSCRIPTS IN MATERNALBLOOD

S Tong*1, C Whitehead1, D Wu2, K Palmer1, J Mockler1, EM Wallace1. 1Centrefor Women’s Health Research, Monash Institute of Medical Research,Australia, 2Bioinformatics Division, Walter and Eliza Hall Institute ofMedical Research, Australia

Introduction: RNA from the fetoplacental unit is released into thematernal circulation, and is remarkably stable. Therefore, it may bepossible to transcriptionally profile the placenta non-invasively bymeasuring RNA in a maternal venepuncture sample. We set out to examinethe profile of mRNA transcripts in maternal whole blood in associationwith severe early onset growth restriction (IUGR), and to determine mRNAchanges with administration of corticosteroids.Methods: We collected maternal whole blood from 4 healthy antenatesand two patients at 27 weeks’ gestation with severe early onset IUGR.We collected further samples from the two patients with IUGR 24hours after administration of corticosteroids (and documented ultra-sound doppler changes in response to the steroids). We used thePaxgene system [PreAnalytix] to collect and isolate the RNA, confirmedpurity [Experion], and did a genome-wide microarray [Illumina plat-form]. We performed differential analyses, which included assessmentof genes grouped according to biological pathways (Gene ontogenyanalysis).

Results: There were 42 genes differentially expressed between the IUGRand healthy antenatal cohort. Corticosteroid administration to the IUGRgroup resulted in very significant changes in mRNA transcripts, with 431genes differentially expressed. The two samples from the ‘IUGR group postcorticosteroids’ showed a gene profile clustering distinctly different to theremaining six samples (two IUGR pre steroids and the four healthy ante-nates – see heatmap/Figure 1).Gene set enrichment analysis showed that hypoxic, apoptosis, oxygentissue binding and placental genes as notable gene pathways thatincreased significantly when corticosteroids were given.Conclusion: Corticosteroid administration in severe onset IUGR is asso-ciated with acute elevation in mRNA transcripts associated with hypoxia,oxygen binding, apoptosis and placental regulation. These changes areconsistent with animal and human observations that corticosteroidadministration to IUGR pregnancies may have detrimental effects. Theseobservations are being currently verified in a larger cohort.

Figure 1. Heat map of 58 representative genes to show IUGR post steroids group (n¼2)cluster differently from IUGR pre-steroids (n¼2) and healthy antenates (n¼4).

Keywords: glucocorticoids, mRNA, hypoxia, microarray


[P04.01].DECIDUAL NEUTROPHILS A NOVEL FINDING: THEIR ROLE IN SECONDTRIMESTER PLACENTATION

HA Amsalem*1,2, CD Dunk1, SL Lye1,2, A Hazan2. 1Samuel LunenfeldResearch Institute, Mount Sinai Hospital, Toronto, Canada, 2University ofToronto, Canada

Objectives: The fetomaternal interface once thought to be an immuneprivilege site is now known to harbour a large population of maternalimmune cells. These cells are crucial to the angiogenesis and vascularremodelling accompanying normal placentation. Accumulating datashows that neutrophils, a major part of the innate immune system, mayplay a crucial role in vascular remodelling within tumor tissue. Little isknown regarding the presence and role of neutrophils in normal andaberrant placentation. We hypothesize that neutrophils infiltrate secondtrimester decidua from the maternal peripheral circulation and contributeto vascular remodelling.Methods: Following informed consent, second trimester decidual tissues(n¼5) were collected from patients during social termination of normalpregnancy. Leukocytes were released from decidual tissues by mechanicalmincing and immunostained for FACS analysis using Anti CD66b and CD15(as neutrophil markers) and CD 45 (leukocytes marker). The same deciduawas also used for immunohistochemistry staining in order to localizea specific cellular distribution of cells within the tissue. Decidua tissueswere immunostained for CD66b, neutrophil elastase (neutrophil markers)and cytokeratine.Results: Our FACS analysis showed that 5-10% of the total CD45+ cells(leukocytes) in the second trimester decidua are neutrophils. NKsubpopulations were used as ‘‘purity control’’ to rule out peripheral blood‘‘contamination’’. Immunostaining confirmed the presence of neutrophilswithin decidual tissue. Neutrophils were observed adhered to endothe-lium and infiltrating the vascular wall. Large aggregates of neutrophilswere seen within the decidual stroma. Our preliminary data suggest thatneutrophil infiltration is restricted to the decidua basalis.Conclusions: This observation is the first demonstration of a residentneutrophil population in the second trimester decidua. Future studies willaddress their potential role at the fetomaternal interface.Keywords: decidua, neutrophils, infiltration, vascular remodelling

[P04.02].DECIDUAL MACROPHAGES FROM FIRST TRIMESTER PREGNANCIES ATINCREASED RISK OF PRE-ECLAMPSIA HAVE LOWER MHC CLASS IIEXPRESSION AND ALTERED CYTOKINE PRODUCTION

C Austen*, AP Johnstone, R Fraser, G StJ Whitley, B Thilaganathan,JE Cartwright. St George’s University of London, United Kingdom

Introduction: Macrophages constitute 20-30% of the decidual immunecells at the site of implantation. The role these cells have in the regulationof placentation or the pathology of pre-eclampsia has not been deter-mined. Measurement of uterine artery resistance indices (RI) by DopplerUltrasound in the first trimester can be used to identify pregnancies witha 30% risk of developing pre-eclampsia (bilateral uterine artery notchingand mean RI>95th centile) or <1% risk (no notches and a mean RI<95thcentile). The aim of this study was to compare first trimester decidualmacrophages isolated from pregnancies screened, prior to termination ofpregnancy, as being at the highest and lowest risk of developing pre-eclampsia, had the pregnancy progressed.Methods: CD14+ macrophages were isolated from first trimester deciduausing antibody-coated magnetic beads. Expression of MHC Class I and IIwas determined by flow cytometry. The secretion of cytokines during 16hculture was assessed using a cytokine array (Proteome Profiler Antibodyarray, R&D Systems).Results: Macrophages from high resistance pregnancies showed reducedexpression of MHC Class II and lower levels of IL-1b, IL-6, MIP-1a and TNFasecretion compared with cells from normal resistance pregnancies. Nodifferences were detected in MHC Class I expression.Discussion: First trimester decidual macrophages from pregnancies clas-sified as at higher risk of developing pre-eclampsia differ in their MHCClass II expression and cytokine secretion compared to lower risk preg-nancies. First trimester decidual macrophages in a lower risk pregnancymay have an increased activation state (indicated by the higher level ofMHC Class II) which, in addition to the higher levels of pro-inflammatorycytokine production, may be important in the promotion of trophoblastinvasion. During a pregnancy with a high resistance index the altereddecidual cytokine environment, contributed in part by the macrophages,may be a factor in the development of pre-eclampsia.Keywords: Macrophage, Pre-eclampsia, MHC, Cytokine


[P04.03].NECROTIC DEPORTED TROPHOBLASTS APPEAR TO ACTIVATEMACROPHAGES: A POTENTIAL MECHANISM CONTRIBUTING TO THEPATHOGENESIS OF PREECLAMPSIA

Q Chen*1,2, JX Ding1, LM Chen1, HY Jin1, PR Stone2, LW Chamley2. 1TheHospital of Obstetrics & Gynaecology, Fudan University, China, 2Depart-ment of Obstetrics & Gynaecology, The University of Auckland, NewZealand

Preeclampsia is characterised by an exaggerated maternal inflammatoryreaction. In normal pregnancy, trophoblasts die by an apoptosis-likeprocess and are shed into the maternal blood then deported from theuterus. In preeclampsia it has been suggested that the shed trophoblastsare more necrotic. It is unclear how shed/deported trophoblasts arecleared from the maternal circulation but macrophages may be involved.Although trophoblasts do not express classical class I HLA, they areimmunologically foreign to maternal immune system. We previouslyreported that phagocytosis of apoptotic trophoblasts by macrophagesresulted in an antiinflammatory/immune suppressing response. Here wecompared the effects of necrotic and apoptotic shed trophoblasts onmacrophages.Red fluorescent-labelled shed trophoblasts were harvested from placentalexplants and either used directly (apoptotic) or induced to undergosecondary necrosis by freeze-thawing. The shed trophoblasts were fed toPMA-treated U937 macrophages activation examined by immunostainingfor HLA-II expression. Peripheral blood mononuclear cells (PBMNCs) werecultured in the presence of supernatants from untreated U937 cells orU937 cells that had phagocytosed apoptotic or necrotic trophoblasts andthe PBMNC proliferation determined by Alarmar Blue assay (Invitrogen).Confocal microscopy demonstrated that HLA-II expression was substan-tially increased on U937 cells that had phagocytosed necrotic shedtrophoblasts compared to U937 cells that had phagocytosed apoptoticshed trophoblasts or untreated controls. The proliferation of PBMNCstreated with conditioned medium from U937 cells that had phagocytosednecrotic shed trophoblasts was significantly greater (p<0.005) than theproliferation of PBMNCs treated with conditioned medium from U937 cellsthat had phagocytosed apoptotic trophoblasts or untreated U937 cells.These data suggest that changing the death process leading to trophoblastshedding towards a more necrotic process could result in an inappropriatematernal immune response initiated by maternal macrophages. Such animmune response might contribute to the exaggerated maternal inflam-matory reaction of preeclampsia.Keywords: trophoblast deportation, HLA-II, marcophages, phagocytosis

[P04.04].CHARACTERIZATION OF ANTIGEN PRESENTING CELL SUBSETS INPREGNANCY-ASSOCIATED MALARIA DURING A FOLLOW-UP STUDY INBENIN

N Fievet*2, S Ibitokou11, B Vianou11, C Agbowaı1, M Oesterholt5,1,A Massougbodji1. 1University of Abomey Calavi, Benin, 2UR010 IRD, Benin,3Institute of International Health, University of Copenhagen, Denmark,4Wenner-Gren Institute, Stockholm University, Sweden, 5RadboudUniversity Nijmegen, Netherlands, 6UR010, IRD, IFR 71 Universite ReneDescartes, France

Introduction: The central phenomenon in the pathogenesis of PregnancyAssociated Malaria (PAM) is the accumulation of P. falciparum (Pf) infectederythrocytes in the placenta. The STOPPAM consortium conducts 2 cohortstudies in pregnant women from Benin and Tanzania to evaluate theimmunopathological consequences of Pf infections during pregnancy.Dendritic Cells (DCs) are of particular relevance in pregnancy-relatedinfections given their role to induce pregnancy tolerance, and antigen-specific immunity, thus contributing in protecting the mother frominfections without compromising fetal survival. Data on DCs in PAM areneeded to understand the cellular mediated immunity (CMI) implicationin a future PAM vaccine design.Methods: In Come, southwestern Benin, a longitudinal prospective studyof 1000 pregnant women is ongoing. Pregnant women are enrolled before24 weeks of pregnancy and followed with full clinical, haematological, andparasitological investigations at each ANV until delivery.CMI is performed 1) in a subgroup of 150 women at inclusion: 75 womenwith active Pf infection, matched for gravidity and gestational age with 75women with neither Pf infection at inclusion nor history of such infectionduring earlier pregnancy; 2) in a subgroup of 120 women at delivery: 40with an active placental Pf infection, 40 with no placental infection buthaving presented with Pf infection during pregnancy, and 40 with no Pfinfection during whole pregnancy. Ex vivo DC and monocyte phenotyping,and activation of antigen presenting cells after LPS activation are evaluatedusing flow cytometry.Results and Discussion: We will compare data on women at inclusion andat delivery according to the timing and pathology of malaria infection.Hitherto, CMI was explored in 100 women at inclusion and in 25 atdelivery. At time of the IFPA congress, all women will have beeninvestigated.Keywords: Plasmodium falciparum, dendritic cells, monocytes, Placenta


[P04.05].DECIDUAL NATURAL KILLER CELLS FROM PREGNANCIES AT HIGHERRISK OF PRE-ECLAMPSIA FAIL TO INDUCE ENDOTHELIAL APOPTOSIS:A DEFECT IN SPIRAL ARTERY REMODELLING?

R Fraser*, GStJ Whitley, AP Johnstone, B Thilaganathan, JE Cartwright.St. George’s, University of London, United Kingdom

Introduction: During pregnancy uterine spiral arteries are remodelledinto larger diameter, higher flow vessels. This change is essential for thedeveloping fetus to obtain sufficient oxygen and nutrients. In pre-eclamptic pregnancies insufficient remodelling occurs. Trophoblasts playa role in spiral artery remodelling through induction of vascular cellapoptosis. However, the role of decidual natural killer cells (dNK), whichare the major maternal immune component of the decidua, has not beendetermined in remodelling. The aim of this study was to establish theeffect of dNK on vascular cells and to investigate whether there are func-tional differences in dNK isolated from pregnancies at higher risk ofdeveloping pre-eclampsia.Methods: Measurement of uterine artery resistance indices (RI) byDoppler Ultrasound in the first trimester can be used to identify preg-nancies with a 30% risk of developing pre-eclampsia (bilateral uterineartery notching and mean RI>95th centile) or <1% risk (no notches anda mean RI<95th centile). CD56+ dNK cells were isolated from firsttrimester decidua by positive selection using antibody-coated magneticbeads. Endothelial cells were co-cultured with dNK cells, the NK92 cell lineor NK cell conditioned medium. Apoptotic morphology was examined bytime-lapse microscopy.Results: Endothelial cells underwent apoptosis in the presence of dNKcells isolated from normal RI pregnancies. This effect was seen in direct co-culture but not in the presence of dNK conditioned medium or when NK92cells were used. Apoptosis was inhibited in the presence of the caspaseinhibitor zVAD-fmk. dNK from high RI pregnancies did not induce endo-thelial cell death above basal levels.Discussion: Decidual NK cells can cause endothelial cell death throughdirect interactions and may therefore have a role in spiral artery remod-elling. The deficiency in endothelial apoptosis induction shown by dNKfrom high RI pregnancies may contribute to the impaired remodelling seenin pre-eclamptic vessels.Keywords: natural killer cell, pre-eclampsia, endothelial cell, apoptosis

[P04.07].AUTOIMMUNITY TO THE ENDOPLASMIC RETICULUM RESIDENTPROTEIN CALRETICULIN DURING HUMAN PREGNANCY

N. M. Gude*1,2, J. L. Stevenson1, P. M. Sheehan1,2, S. P. Brennecke1,2. 1RoyalWomen’s Hospital, Australia, 2University of Melbourne, Australia

The calcium-binding endoplasmic reticulum resident protein calreticulinis significantly increased in maternal blood throughout human pregnancycompared to the non pregnant state (Gu et al, Molec Hum Reprod, 14:309-315, 2008). The role of circulating calreticulin in human pregnancy is notknown. Calreticulin can generate an autoimmune response when presentin the extracellular environment. This has been proposed to contribute tothe progress of autoimmune diseases such as systemic lupus eryth-ematosus. The aim of this study was to determine the prevalence of anti-calreticulin IgG in a cohort of pregnant women, and to assess changes inantibody titre throughout gestation. 103 women without autoimmunedisease or other pre-existing pathology were recruited at their firstantenatal visit and blood was taken at approximately fortnightly inter-vals. Autoantibodies were measured using a high stringency ELISA andtitrated against a standard source of anti-calreticulin IgG. A positivereaction was taken as >mean plus 3 standard deviations of the non-specific binding wells. Pregnancy outcomes were: 85 normal, 6 pre-eclampsia (2 with fetal growth restriction), 5 pregnancy-induced hyper-tension, 4 gestational diabetes, 2 preterm labour and 1 fetal growthrestriction alone.

This is the first time anti-calreticulin autoantibodies have been reported inhuman pregnancy. The prevalence in this cohort of pregnant women (5.8%)is similar to that observed by other studies for non pregnant, controlpopulations (i.e. without autoimmune disease). Further work is required todetermine if the presence of maternal autoantibodies to calreticulin duringpregnancy is associated with altered risk of adverse pregnancy outcome.Keywords: calreticulin, autoimmunity, maternal blood, antibody


[P04.08].CD4+FOXP3+ T REGULATORY CELLS ABUNDANCE DURING PREGNANCYIS INFLUENCED BY INTERLEUKIN-10 IN CONCERT WITH FETALALLOANTIGENS

LR Guerin*1, JR Prins2, JD Hayball3, SA Robertson1. 1Research Centre forReproductive Health, Discipline of Obstetrics and Gynaecology, Universityof Adelaide, Australia, 2Department of Obstetrics and Gynaecology,University Medical Center Groningen, University of Groningen,Netherlands, 3Hanson Institute and Sansom Institute, Australia

Maternal immune tolerance of conceptus alloantigens is critical inensuring pregnancy success. T cells known as regulatory T cells (Treg cells)that express the hallmark markers CD4 and Foxp3 are essential formediating fetal tolerance. However, the factors that control Treg cellsduring pregnancy are unknown. We endeavoured to analyse the role ofinterleukin-10 (IL-10) in regulating Treg cell function and abundance inpregnancy.Female IL-10 deficient (IL-10-/-) or wild type (WT) C57Bl/6 were matedwith either Balb/c (allogeneic) or C57Bl/6 (syngeneic) males. Treg cellswere analysed using anti-Foxp3 antibodies and FACS analysis in the iliacand inguinal lymph nodes or in the uterus via immunohistochemistrythroughout gestation.IL-10 deficiency was shown to result in an elevation of the percentage ofCD4+ cells expressing the Treg cell marker Foxp3 by approximately 30%,and to increase total cell numbers by w2-fold in all lymphoid tissuesanalysed. In females gestating allogeneic foetuses, there was an increase inTreg cells in the uterine draining (iliac) lymph nodes, but not other lymphnodes, with a peak of w10-fold more Treg cells at gestational day (gd) 10.This was accompanied by an w50% increase in the percentage of CD4+ cellsexpressing Foxp3 in both the iliac and inguinal lymph nodes. In micegestating syngeneic fetuses there was no increase in the percentage ofCD4+ cells expressing Foxp3 due to IL-10 deficiency. When CD4+ Foxp3+ Tcells were recovered from iliac lymph nodes of pregnant females andanalysed in mixed lymphocyte reaction assays using paternal lymphocytesin vitro, there was no effect of IL-10 genotype on suppressive function.These findings highlight an important role for IL-10 in Treg cell abundanceand lineage commitment throughout gestation. Additionally they indicatea role for fetal alloantigens in promoting the conversion ofCD4+ cells toFoxp3-expressing Treg cells.Keywords: Treg, interleukin 10

[P04.09].COLOCALIZATION OF HLA-G5, B7-H1 AND LAMP-1 PROTEINS IN THEHUMAN PLACENTA

S.K. Kshirsagar, A.S. Trikhacheva, M.G. Petroff*. University of KansasMedical Center, Kansas, United States

The semi-allogeneic fetus enjoys immune privilege by promotingmaternal-fetal immunological tolerance. The outermost trophoblast-derived layers of the placenta maintain this status by forming a physicalbarrier with immunomodulatory properties between the mother and thefetus. Among the immunosuppressive proteins expressed by these cells areB7-H1 and HLA-G. In addition to being expressed on the cell surface, theseproteins have been found to be associated with trophoblast-derived exo-somes in the maternal circulation, suggesting a role for these factors insystemic modulation of the maternal immune system. The aim of thisstudy was to determine whether placental B7-H1 and HLA-G proteins areassociated in situ with the secretory lysosomal pathway, which can lead toexosome secretion. The expression of B7-H1, HLA-G and Lamp-1 inplacental tissue from term and first trimester placenta was examined byimmunohistochemistry and immunofluorescence microscopy. In the syn-cytiotrophoblast and extravillous trophoblast, B7-H1 and Lamp-1 colo-calized at the maternal-fetal interface. Using the isoform specific antibody12C3, HLA-G5 expression was observed in villous cytotrophoblast cells andextravillous trophoblast cells. Similar staining patterns were observed infirst trimester and term placentas. Cytospin preparations of the termtrophoblast cells clearly showed colocalization of Lamp-1 and HLA-G5 ina punctate, intracellular pattern. Finally, HLA-G5 and Lamp-1, but not B7-H1, also colocalized in the placental Hofbauer cells. Our results suggest thatB7-H1 and HLA-G proteins are associated with secretory lysosomalpathway in the placenta, and support the hypothesis that they can besecreted via exosomes. This work was supported by NIH grants HD045611and HD049480.


[P04.10].FETAL ANTIGEN INDUCES TOLERANCE IN MATERNAL CD4+ T CELLS

A. Perchellet, M.G. Petroff*. University of Kansas Medical Center, Kansas,United States

Tolerance of the maternal immune system is believed to be important forsuccessful pregnancy as the fetus is semi-allogeneic and may be subject toanti-fetal responses. We examined maternal T cell tolerance to the fetus inmice using a system in which a model antigen, ovalbumin (OVA), isexpressed exclusively in the fetus. This is achieved by breeding femalesthat lack OVA to transgenic males that ubiquitously express membrane-bound OVA. By employing T cell receptor (TCR) transgenic mice specific foreither a MHC class I or class II-restricted epitope of OVA (OT-I and OT-II,respectively) as mothers, we investigated the fate of fetus-specific CD8+

and CD4+ T cells during gestation. Both CD8+ and CD4+ T cells displayed anactivated phenotype in the spleen and lymph nodes of OVA-bred OT-I andOT-II mice, indicating their encounter with fetal antigen in lymphoidtissues. A small percentage of CD4+ T cells were deleted in the peripheryand thymus of OVA-bred OT-II mice, with evidence of TCR downregulationin the remaining T cells. However, deletion and TCR downregulation werenot observed in OVA-bred OT-I mice. Both CD4+ and CD8+ T cells upregu-lated ICOS expression in the presence of specific fetal antigen, but onlyCD4+ T cells consistently upregulated the inhibitory receptors PD-1 andCTLA-4. More regulatory T cells were present in OVA-bred than in WT-bredOT-II mice, suggesting that fetal antigen specifically stimulates expansionof these cells. These data indicate that fetal antigen-specific maternal CD4+

T cells are tolerized during gestation by several potential mechanisms,whereas tolerance of fetal antigen-specific CD8+ T cells is less effective. Thisnotion is supported by the observation that fetal loss occurred in OVA-bredOT-I, but not OT-II, mice. This project is supported by NIH grants HD045611and HD049480. A. Perchellet is supported by NIH training grantT32HD007455.

[P04.11].MINOR HISTOCOMPATIBILITY ANTIGEN EXPRESSION IN TROPHOBLASTAND FETAL BLOOD CELLS: IMPLICATIONS FOR MATERNAL-FETALIMMUNE TOLERANCE

M.G. Petroff*, K.M. Adams-Waldorf, J. Zhao. University of Kansas MedicalCenter, Kansas, United States

Pregnancy represents a unique physiological situation in which a motherestablishes robust immunological tolerance to the semiallogeneic fetus.Increasingly, there is evidence that this tolerance includes accommodationby antigen-specific lymphocytes to specific paternally-inherited alloanti-gens. Murine transgenic systems have shown specific activation, prolifer-ation, and deletion of fetal antigen-specific T cells, and in women,expanded cohorts of fetal antigen-specific T cells are frequently detected asa result of pregnancy. We have previously hypothesized that antigensderived from the syncytiotrophoblast and fetal blood cells access maternalantigen presenting cells and tolerize lymphocytes by way of trophoblastshedding and fetal microchimerism, respectively. Here, we address thisquestion by asking whether the placenta and fetal blood leukocytes couldbe a source of antigens in human pregnancy, with a focus on two distinctminor histocompatibility antigens (mHAg): the autosomally-encodedmHAg HA-8, and the Y chromosome-encoded mHAg SMCY. RT-PCR anal-ysis of whole placenta lysate as well as purified trophoblast cells revealedthat both HA-8 and SMCY mRNA are expressed in placenta and trophoblastcells. SMCY was detected only in male placenta and trophoblast samples;placentas and trophoblast cells from female infants failed to yield an RNAproduct. Purified fetal cord blood leukocytes also expressed RNA for bothmHAg. Finally, HA-8 protein was found by immunohistochemistry to bepresent within the cytoplasm of the syncytiotrophoblast of term placentas.In the basal plate placenta, HA-8 antibody also reacted lightly withextravillous trophoblast cells. These results provide new evidence thattrophoblast cells and fetal blood leukocytes are a source of proteins thatcould be antigenic to maternal leukocytes, and support the hypothesis thatmaternal lymphocytes could recognize these fetal antigens if presented inthe context of maternal antigen presenting cells. This work was supportedby NIH grants HD045611 and HD049480.


[P04.12].GENE EXPRESSION OF TOLL-LIKE RECEPTORS (1–10) IN FIRSTTRIMESTER TROPHOBLASTS, AS ASSESSED BY RT-qPCR ANALYSIS

G.D. Olsen*, A.S. Gundersen, A-H. Leknes, T.D. Nguyen, R. Austgulen,A-C. Iversen. NTNU, Norway

Introduction: The placenta constitutes a physical and immunologicalbarrier to protect the fetus against invading infectious agents andinflammation. So far, local maternal immune cells have been assigned themost active immunological role, but a number of observations, such as thedocumentation of Toll-like Receptor (TLR) gene expression, suggest thatfetal trophoblasts contribute to placental immunity and inflammatoryresponses.The aim of this study was to further explore the gene expression of TLRs introphoblasts to extend knowledge of relevance for an immunological role.Gene expression of all TLRs in first trimester trophoblasts was quantifiedand compared to corresponding findings in fibroblasts and trophoblast celllines.Methods: Primary trophoblasts and fibroblasts were isolated from firsttrimester placentas (5-12 gestational weeks). The choriocarcinoma celllines JEG3 and JAR and normal human dermal fibroblasts (NHDF) wereincluded for comparison. Gene expression of TLR 1-10 was analyzed in allcell types by quantitative real time reverse transcription PCR (RT-qPCR).Results/Discussion: Interestingly, first trimester trophoblasts demon-strated a relatively high expression of most TLRs, more pronounced thanthat observed in both the trophoblast cell lines and the different fibro-blasts. A more restricted TLR expression level was observed in primaryfibroblasts than in NHDF, whereas in trophoblast cell lines, JEG3 showedlower TLR expression levels than JAR.Furthermore, substantial variations in TLR gene expression levels wereobserved between individuals in first trimester trophoblasts.Conclusion: The pronounced TLR gene expression in primary trophoblastsis rather suggestive of an active immunological role during placentation.Further studies on TLR protein expression and function in these tropho-blasts are warranted.Keywords: Trophoblasts, Inflammation, Toll-like receptors, RT-qPCR

[P04.13].HUMAN DECIDUAL TISSUE CONTAINS DISARMED CD8+ EFFECTOR-MEMORY T CELLS

T Tilburgs*1,2, CMC Schonkeren1, M Eijkmans1, DL Roelen1, SA Scherjon1,FHJ Claas1. 1Leiden University Medical Center, Netherlands, 2HarvardUniversity, United States

During pregnancy maternal lymphocytes at the fetal-maternal interfaceplay a key role in the immune acceptance of the allogeneic fetus. DecidualNK cells contain immune modulatory properties and facilitate trophoblastinvasion into maternal tissue. More recently, CD4+CD25bright regulatory Tcells have shown to be concentrated in decidual tissue where they are ableto suppress fetus-specific and non-specific responses. However, decidualCD8+ T cells form the largest fraction of T cells at the fetal-maternalinterface but limited data is present on the characteristics of these cells.Therefore we performed phenotypic analysis of the decidual and periph-eral CD8+ T cell pool with CD45RA, CCR7, CD28 and CD27 expression usingnine-colour flowcytometry. In addition, we examined expression of thecytolytic molecules perforin, granzyme B and granzyme K to determine thecytotoxic potential of the decidual CD8+ T cell subsets. Our data demon-strate that decidual CD8+ T cells mainly consist of differentiated Effector-Memory cells while unprimed naıve cells are almost absent. Unlikeperipheral blood Effector-Memory CD8+ T cells, the decidual Effector-Memory CD8+ T cells do not express perforin and have a reducedexpression of granzyme B. Apparently, the functional features of decidualCD8+ T cells do not correspond their matching phenotype in peripheralblood. These data show that decidual CD8+ T cells may pursue alternativemeans of effector cell differentiation and indicate that decidual CD8+ T celldifferentiation and regulation may play a crucial role in maternal immunetolerance to the fetus.Keywords: Decidua, CD8+ T cells, Effector-Memory differentiation, Human

[P04.14].CD11c IDENTIFIES TWO DISTINCT SUBSETS OF DECIDUALMACROPHAGES

BL Houser, ML Nicotra, T Tilburgs*, JL Strominger. Harvard University,United States

Placentation is a defining characteristic of Eutherian mammals. However,how the maternal immune system tolerates the fetal allograft duringhuman pregnancy remains unclear. After NK cells, macrophages comprisethe second largest leukocyte population in the decidua, at 20-25% of allimmune cells. Here we demonstrate that there are two distinct subsets ofdecidual macrophages based on CD11c expression (CD11cHI and CD11cLO).Approximately, 35% of decidual macrophages are CD11cHI and 65% areCD11cLO. These two populations have unique cell morphologies andspecific surface markers. Gene expression analysis by RNA microarrayrevealed over 200 genes that were differentially expressed between thesetwo populations. Functional analysis revealed that the two subsets do notfunctionally differ in their phagocytic capacity. However, compared to invitro derived macrophages, both decidual macrophage populations are lessphagocytic. These data show that two previously undescribed subsets ofmacrophages are found in the decidua.Keywords: Decidua, Macrophages, Human, CD11c


[P05.03].EPIDERMAL GROWTH FACTOR (EGF) STIMULATES UPREGULATION OFMMP-9 AND TIMP-1 IN BOVINE PLACENTAL CELLS VIA MAPKSIGNALLING PATHWAY

Marc Dilly*, Nina Hambruch, Jan Dirk Haeger, Christiane Pfarrer. Depart-ment of Anatomy, University of Veterinary Medicine, Hannover, Germany

The bovine synepitheliochorial placenta is characterized by restrictedtrophoblast invasion, a unique feature of which the regulatory mecha-nisms are not yet fully understood. Among other factors, matrix metal-loproteinases (MMPs) and counteracting tissue inhibitors ofmetalloproteinases (TIMPs) serve to control cell migration and tissueremodelling. MMP-9 is present in the bovine placenta throughout gesta-tion; its proteolysis is predominantly regulated by the action of endoge-nous TIMP-1. Epidermal growth factor (EGF), as regulator of fundamentalcell properties, is capable to up-regulate MMP-9 activity in a variety of cellstypes. However, there is no direct evidence for the stimulation of MMP-9and TIMP-1 expression by EGF in the bovine placenta. In addition, thesignalling pathways involved in this regulation are not clear.In this in vitro study, cultured maternal caruncular epithelial cells (BCEC-1)and trophoblast cells (F3) isolated from bovine placenta were used toexamine the possible involvement of the mitogen-activated protein kinase(MAPK) pathway in the regulation of the MMP-9/TIMP-1 system by EGFand MAPK inhibitor application. The activity of MAPK was determined byWestern blot while mRNA levels of MMP-9/TIMP-1 were compared bydensitometric analysis of specific RT-PCR products. MMP activity wasanalyzed by zymography.We demonstrated that EGF increases both MMP-9 and TIMP-1 mRNAexpression in BCEC-1 and F3 cells. This effect could be abolished byinhibiting MAPK activation. In the same time, Western analysis showeda tremendous activation of MAPK exclusively in F3 cells, whereaszymography revealed that EGF elevates MMP-9 activity predominantly inBCEC-1 cells.The results presented suggest that EGF activates the MAPK pathway inbovine placenta cells, and this activation is necessary for the up-regulationof MMP-9 and TIMP-1 expression. Thus EGF may be involved in theregulation of restricted trophoblast invasion and defined tissue remodel-ling during bovine gestation.Funded by the German Research Foundation (DFG).Keywords: Bovine, EGF, MMPs, MAPK

[P05.04].ACTIVIN A REGULATES HUMAN EXTRAVILLOUS TROPHOBLAST CELLINTEGRINS AND ADHESION TO EXTRACELLULAR MATRIX

E Dimitriadis*, C Stoikos, LA Salamonsen. Prince Henry’s Institute ofMedical research, Australia

Introduction: Successful pregnancy depends on adequate invasion ofextravillous trophoblast (EVT) into the uterine decidua. Locally producedactivin A has been proposed to have a role in EVT invasion however themechanisms by which this occurs are poorly understood. We hypothe-sized that activin A regulates trophoblast invasion by modulating EVTadhesive properties. This study investigated whether activin A influenceshuman EVT integrin molecule production and EVT adhesion to variousECM.Methods: The human EVT cell line, HTR-8/SVneo (HTR8) was used asa model for human EVT. The expression of activin receptors (R) ActivinR1a,ALK4 and ActivinRIIA/B on HTR8 cells was examined by RT-PCR. The effectof activin A on HTR8 integrin molecule expression was assessed by integrinantibody arrays while activin A’s role on trophoblast cell adhesion tofibronectin (FN), collagen (COL) 1, COLIV, vitronectin (VN) and laminin (LN)was measured by cell-matrix adhesion assays. The effect of activin A onphosphorylated (p) and total SMAD abundance in HTR8 cells was assessedby Western blot.Results: HTR8 cells expressed ActivinR1a, ALK4 and ActivinRIIA/B mRNA.Activin A (1, 10, 50, 100 ng/ml) dose dependently increased pSMAD2abundance while SMAD2 was unaffected in HTR8 cells. The activininhibitor, SB431542 (SB: 10mM) abolished pSMAD2 protein abundance butpSMAD2 increased when SB was added with activin A (50 ng/ml)compared to SB alone. HTR8 cells adhered maximally to FN, COL1 andCOLIV. Activin A (50ng/ml for 24h) decreased cell binding to fibronectin,COLI and COLIV (p<0.05), and cell surface integrin subunits a1 a2 a3 a5, b1,b2 and b4 (p<0.05) in HTR8 cells.Conclusion: This is the first study to demonstrate that activin A regulateshuman trophoblast cell surface adhesion molecule production and adhe-sion to various ECM. This suggests a mechanism by which activin Aregulates trophoblast cell invasion during early pregnancy.Keywords: trophoblast adhesion, activin a, integrins, trophoblast invasion


[P05.05].ASPARTYL-ASPARAGINYL b-HYDROXYLASE: A GENE CRITICAL FORPLACENTATION AND FETAL GROWTH

F Gundogan*1,2, AD Bedoya2, ES Lau2, P Mark3, JR Wands2,3, SM de laMonte2,3. 1Women and Infants Hospital, United States, 2Brown AlpertMedical School, United States, 3Rhode Island Hospital, United States

Introduction: Aspartyl-asparaginyl b-hydroxylase (AAH) is a type 2transmembrane protein that hydroxylates epidermal growth factor-likedomains of Notch and Jagged, which have known roles in cell migrationand invasion. Placenta is the only non-transformed organ that expresseshigh levels of AAH, and extravillous cytotrophoblast, which are motile andinvasive, express higher levels of AAH than the non-motile villous coun-terpart. In humans, impaired placentation accompanied by intrauterinegrowth restriction (IUGR) was found to be associated with reduced levelsof AAH in extravillous trophoblast. In the present study, we directlyaddressed the roles of AAH in placentation and trophoblast motility usingin vitro and in vivo gene silencing approaches.Methods: HTR-8 SVneo human trophoblastic cells were transfected withsiRNA targeting AAH (siAAH) or no specific sequences (siScr) using theAmaxa electroporation system. Directional motility was measured usingthe ATP luminescence based motility and invasion assay. To study theeffects of siAAH in vivo, pregnant Long Evans dams were anesthetizedand subjected to laparotomy to microinject siAAH or siScr directly intothe mesometrial triangle on gestational day 17. The siRNA molecules wereco-transfected with GFP expressing plasmids to monitor delivery andtransfection efficiency. Placentas harvested 24 h later, were used tomeasure AAH, Notch, Jagged and HES (downstream target of Notch) byqRT-PCR.Results: SiRNA silencing of AAH significantly reduced the mean directionalmotility in HTR-8 SVneo trophoblastic cells relative to siScr transfectedcontrol cells. In vivo intra-placental delivery of siAAH was successfullyaccomplished based on the robust GFP fluorescence and demonstration ofreduced AAH mRNA levels by qRT-PCR analysis. Correspondingly, inhibi-tion of AAH expression was associated with significant reductions inNotch, Jagged, and HES-1 mRNA levels, and significant IUGR of the pups(1.46�0.02 g. versus 1.77�0.04 g. in control, P<0.0001).Conclusions: AAH has a critical role in mediating trophoblast motility,which is required for placentation. Inhibition of AAH expression, such asthat caused by maternal consumption of alcohol, leads to impairedplacentation and intrauterine growth restriction. Therapeutic measures tosupport or bolster AAH expression may help reduce the risk of IUGR.Keywords: AAH, motility, placentation, IUGR

[P05.06].BOVINE TROPHOBLAST CELLS INVADE COLLAGEN GELS FROMEMBEDDED SPHEROIDS

Jan Dirk Haeger*, Nina Hambruch, Marc Dilly, Christiane Pfarrer. Depart-ment of Anatomy, University of Veterinary Medicine, Hannover, Germany

Introduction: To overcome the limitations of two-dimensional cell culturesystems, three-dimensional spheroids which are considered to be morelike in vivo have been developed in the past. Spheroids have been formedwith equine chorionic girdle cells and human cytotrophoblasts.Objective: We aimed to generate spheroids with bovine placental tropho-blast cells and to show that these spheroids are suitable to test the influenceof growth factors on the invasion of bovine trophoblast cells in vitro.Methods: Trophoblast cells were seeded in drops on dishes which wereturned upside-down and incubated (hanging drop). Each drop consisted ofa defined cell number, 25% methocoel, matrigel (0.8-1%) and regularculture media. Spheroids were harvested and characterized morphologi-cally by light-, transmission and scanning electron microscopy (LM/TEM/SEM). To determine the viability of spheroidal trophoblast cells spheroidswere incubated with calcein-AM and ethidium-homodimer-1. Addition-ally, spheroids were harvested during formation (1, 2 and 3 days afterseeding) and the nuclei were stained using Bisbenzimid to detect frag-mentation of cell nuclei (IF). For in vitro invasion assays trophoblastspheroids were embedded in collagen gels which were overlayed withserum free media containing EGF (50 ng/ml).Results: Bovine trophoblast spheroids were clearly delimited and coveredby extracellular matrix (LM/SEM). Cells contributing to spheroids wereindiscriminable from each other (LM). The outer spheroidal layer consistedof differentiated cells possessing an apical pole directed to the outside (LM/TEM). The inner core of the spheroids contained degenerating cells whilethe cells of the outer rim were viable (TEM/IF). Spheroidal trophoblast cellsinvaded the collagen gels. EGF strongly enhanced this process.Conclusion: Using bovine trophoblast cells we have generated spheroidsthat are useful to further examine the influence of growth factors on theinvasion of bovine trophoblast cells in vitro and the underlying signallingpathways. Funded by the German Research Foundation (DFG).Keywords: bovine, trophoblast cells, spheroids, EGF


[P05.07].MIGRATION AND DIFFERENTIATION OF A BOVINE TROPHOBLAST CELLLINE IS STIMULATED BY EPIDERMAL GROWTH FACTOR (EGF) INA CONCENTRATION-DEPENDENT WAY

N. Hambruch*, M. Dilly, J.-D. Haeger, C. Pfarrer. Department of Anatomy,University of Veterinary Medicine, Hannover, Germany

In bovine placentomes, trophoblast giant cells (TGC), differentiating fromuninucleate trophoblast cells (UTC), are crucial for feto-maternal inter-actions as they have the unique ability to migrate into and fuse with thematernal epithelium. Since this differentiation and migration is essentialfor successful gestation, we established a bovine trophoblast cell line(F3) to investigate the influence of potentially regulatory factors on themigratory activity in vitro. One likely candidate is epidermal growthfactor (EGF) which is known to regulate fundamental cell properties,such as growth, differentiation and invasion. Consequently, we aimed tostudy EGF mediated effects on the regulation of F3 cellular growth,differentiation and motility with special respect to different EGFconcentrations.F3 cells were incubated with EGF in concentrations ranging from 10 to100ng/mL in serum free media for 24h. Subsequently, cell proliferation wasmeasured by colorimetric MTT assay. Additionally, a wound-healing assay(motility assay) was carried out to analyse the influence of EGF on themigratory activity. Furthermore, the ratio of binucleate TGC to UTC wasdetermined after stimulation with 10 or 100ng/ml EGF.Incubation of F3 with EGF led to a significant enhancement (p<0,001) inproliferation compared to serum deprived cells, with highest mitogenicresponses at 10 and 50ng/ml, whereas 100ng/ml showed the weakesteffect. Consistent with these results the stimulation with 10ng/ml EGFresulted in a higher motility than with 100ng/ml as shown by wound-healing assay. When comparing the effect of EGF on the ratio of binucleateTGC to UTC first experiments indicated that high EGF concentrationsfavour the formation of TGC.In conclusion, our data demonstrate that EGF is able to stimulate growthand motility of bovine trophoblast cells in a concentration-dependentmanner. Furthermore, first results indicate, that EGF may play a role in theregulation of TGC differentiation.Funded by German Research Foundation (DFG).Keywords: Bovine, Trophoblast, EGF-signalling, Cell culture

[P05.08].EXPRESSION OF N-ACETYLGLUCOSAMINYLTRANSFERASE V ISDOWNREGULATED BY TRANSFORMING GROWTH FACTOR b1 ANDHYPOXIA IN EXTRAVILLOUS TROPHOBLAST (EVT) CELLS

K Hayashi*, E Yamamoto, K Ino, K Niimi, S Kondo, F Kikkawa. NagoyaUniversity, Japan

Objectives: N-Acetylglucosaminyltransferase V (GnT-V) is one of the mostrelevant glycosyltransferases to tumor invasion and metastasis, and catalyzesb1-6 GlcNAc branching on N-glycans. We have shown that GnT-V regulatedEVT invasion through glycosylation of a5b1 integrin and GnT-V expressionwasdownregulated in EVTs invading the decidua. In the present study, we inves-tigate the molecular mechanisms involved in regulation of GnT-V expression.Methods: 1. We cultured HTR-8/SVneo (EVT cell line) and under normoxia(20%) and hypoxia (1%) for 24hr and 48hr. The effect of hypoxia on GnT-Vexpression was investigated by Western blot analysis and RT-PCR. 2. GnT-Vexpression level was examined in the cell lines with supernatant ofdecidual tissue culture for 48hr, RT-PCR and Western blot were performed.3. To clarify cytokines in decidua involved in regulation of GnT-V expres-sion, we cultured the two cell lines with TGF-b1, TNFa, INF-g, IL-6 for 48hrand RT-PCR and Western blot were performed.Results: 1. The expression of GnT-V in HTR8/SVneo was lower underhypoxia than normoxia by western blot analysis and RT-PCR. 2. The level ofGnT-V expression was decreased by addition of supernatant fluid ofdecidual culture. 3. TGF-b1 significantly decreased the level of mRNA andprotein of GnT-V in a dose-dependent response in HTR-8/SVneo.Conclusion: These results suggest that hypoxia and TGF-b1 regulate thelevel of GnT-V expression and this mechanism may be involved in regu-lation of EVT invasion.Keywords: EVT, GnT-V, TGFbeta1, hypoxia

[P05.09].ESTRADIOL MODULATES THE EXPRESSION OF CHEMOKINE RECEPTORSON A HUMAN MAST CELL LINE AND PROMOTES THEIR MIGRATION TOTHE UTERUS

F Jensen*, A Teles, M Woudwyk, AC Zenclussen. Experimental Obstetrics &Gynecology, Medical Faculty, Otto-von-Guericke University, Magdebug.,Germany

Human embryo implantation is a complex process involving blastocystattachment to the endometrial epithelium and trophoblast invasion.Uterine histamine is a key regulator of implantation due to its capacity ofaltering vascular permeability. As histamine is produced by mast cells(MCs) which are present in the uterus as well as at the fetal-maternalinterface during pregnancy, we aimed to analyze MC migration totrophoblast cells by using a two chamber in vitro system. Since it is knownthat CCL11, CCL14, CCL16 and CCL22 are expressed in the uterus throughoutthe menstrual cycle, and their expression level fluctuate under hormonalinfluence, we further analyzed the effect of estradiol on the expression oftheir receptors in MCs as a possible mechanism for MC migration.We employed the well-characterized human MC line HMC-1. We firstanalyzed the migratory capacity of HMC-1 towards primary humantrophoblasts of first trimester and towards choriocharcinoma cells (JEG-3cell line). HCM-1 cells were further incubated with physiological concen-trations of estradiol and MC degranulation as well as the expression ofCCR4, CCR5 and CXCR4 protein and mRNA were analyzed.We confirmed that MCs strongly migrate to both human primarytrophoblasts and JEG-3 cells. Physiological concentrations of estradiol leadto MC degranulation while significantly up-regulating the expression ofCCR4, CCR5 and CXCR4 in HMC-1 cells.The modulatory effects of estradiol on the expression of chemokinereceptors clearly brings to ligth a novel mechanism as to how MCs maymigrate to the uterus/fetal-maternal interface. Because MCs degranulationproducts as e.g. histamine, are key regulators in implantation, we specu-late that MCs are recruited to the uterus by estrogen influence via up-regulation of chemokines receptor expression, while their degranulationmay prepare the uterus for a possible implantation.Keywords: Mast cells, Chemokines, Trophoblast


[P05.10].VASCULAR-DERIVED CHEMOKINES PROMOTE TROPHOBLAST INVASION

RJ Keogh*1,2, S Chau1,2, M Grgurinovic2, P Murthi2, S Rogerson2,S Brennecke1,2. 1Royal Women’s Hospital, Australia, 2University ofMelbourne, Australia

Introduction: There is a dynamic interaction between trophoblast andvascular smooth muscle cells (VSMC) during the remodelling of uterinespiral arteries in the first trimester of human pregnancy. Characterizationof this interaction shows that movement of trophoblast is directional withgreater persistence and speed in the presence of VSMC (1). This directedmovement of trophoblast is likely to be regulated by chemokines. Che-mokines are a subgroup of cytokines that have an ability to causechemotaxis (directed movement) of nearby responsive cells. In addition toacting as chemo-attractants, chemokines also have other importantfunctions including facilitating cell adhesion, proliferation and survival.VSMC produce chemokines which have a direct role in vascular remod-elling and trophoblast express receptors for a number of different che-mokines, including those made by VSMC.Methods: We have screened arterial SMC for chemokine production usinga protein profile array. Candidate chemokines, for which trophoblastexpress receptors, were identified and their effects on trophoblast functionwere tested using HTR8/SVneo cells as a model of invasive extravilloustrophoblast. Wound healing assays were used to assess effects on migra-tion and gelatin zymography was performed to assess matrix metal-loproteinase activity as an indicator of invasive potential.Results: Four candidate chemokines were identified all of which have thepotential to control trophoblast cell functions. Three of these chemokineshave been tested thus far (CXCL1, CCL2, CCL5). All three chemokines werefound to stimulate trophoblast cell migration in a concentration-depen-dent manner. In addition, all three chemokines were found to up-regulatematrix metalloproteinase 2 and 9 activity. Ongoing work will examine theeffects of these chemokines on trophoblast proliferation.Discussion: We conclude that CXCL1, CCL2, and CCL5 are important factorscontributing to the control of directed trophoblast movement into uterinespiral arteries.Reference(1) Hamzic, E. et al. Experimental Cell Research (2008) 314, 1455-64.Keywords: Chemokine, Trophoblast invasion, Vessel

[P05.11].RCHO-1 TROPHOBLAST MODEL SYSTEM TO STUDY THE EXPRESSIONAND FUNCTION OF HIGH TEMPERATURE REQUIREMENT FACTOR A1

F Ajayi, P Samuels, DA Kniss*. Ohio State University, United States

Preeclampsia continues to be a worldwide complication of human preg-nancy and in the US the incidence is 5-8%. Theories as to the pathogenesisof the disease suggest that inappropriately shallow invasion of extravillustrophoblasts into the uterine spiral arterioles contributes to the failure toestablish a low resistance circulatory environment, and heralds thepathobiology that ultimately gives rise to the clinical symptoms ofpreeclampsia. Moreover, abberant vascular remodeling within theuteroplacental circulation creates a state of relative local hypoxia thatinduces further cellular pathophysiological sequelae. Recent work by ourgroup and by others has demonstrated that High temperature requirementfactor A1 (Htra1, a 55-kDa serine protease) may play a role in placentation,in particular the migratory and invasive phases by the trophoblast.Patients who developed early-onset, severe preeclampsia had elevatedlevels of circulating Htra1 (Ajayi et al., work in progress). In addition,ectopic expression of Htra1 using an in vitro model of extravillustrophoblast (HTR-8 SV/neo) inhibited cell migration and invasion inconventional Matrigel assays (Ajayi et al., AJOG, 2008). Using a ratchoriocarcinoma model of trophoblast (RCHO-1, kind gift from MichaelSoares) we conducted experimental studies to examine the ontogeny ofHtra1 expression as cells differentiated from trophoblast stem cells intotrophoblast giant cells. The expression of Htra1 mRNA (measured by real-time RT-PCR, qRTPCR) and its cognate protein (measured by immuno-blotting and immunofluorescence) was low to absent in cells grown inmaintenance medium, but was substantially up-regulated within 24 hfollowing the transition to differentiation medium. Importantly, therewas a concomitant diminution in Id2 (stem cell marker) and increase inCSH1 (differentiation marker) during differentiation of trophoblast stemcells into multinucleated giant cells. Preliminary studies indicated alsothat hypoxia in vitro was associated with increased Htra1 expression.These early data provide a readily testable model system in which toprobe the biology of Htra1 expression and function in the early stages ofextravillus trophoblast development. (This work was supported in part byPerinatal Resources, Inc., and The Ohio State University Perinatal Researchand Development Fund.)*Maternal-Fetal Medicine Fellow-in-Training.Keywords: Preeclampsia, Trophoblast stem cells, Htra1, Trophoblastinvasion


[P05.12].THE ROLE OF AKT ISOFORMS IN HUMAN TROPHOBLAST MIGRATION

P Haslinger*, S Sonderegger, J Otten, K Biadasiewicz, M Knofler.Department of Obstetrics and Fetal-Maternal Medicine, Austria

Objective: The protein kinase AKT is a well-known regulator of diversecellular processes including proliferation, migration, differentiation andsurvival. With respect to the placenta AKT was shown to be a criticallyinvolved in basal and growth factor-dependent trophoblast migration.However, three different isoforms of AKT exist which, depending on thebiological process, may have agonistic or antagonistic functions. Hence, theaim of this study was to analyse expression of AKT1, AKT2 and AKT3 indifferent trophoblast model systems and to investigate their individualroles in trophoblast proliferation and migration.Methods: Expression of AKT1, AKT2 and AKT3 was analysed by real-timePCR and Western blotting (isoform-specific antibodies) using wholetissues of first and third trimester placentae, purified first and thirdtrimester trophoblasts/stromal fibroblasts, first trimester villous explantcultures, purified EVT as well as trophoblastic cell lines (JEG-3, HTR-8/SVneo, SGHPL-5). Constitutive, stable (puromycin-selected) knock-downcell pools of AKT1, AKT2 and AKT3 were generated in SGHPL-5 cells bytransduction with retroviral vectors (LMP) expressing shRNAmir (micro-RNA-adapted short hairpin RNA) against human AKT1, AKT2 and AKT3,respectively (OpenBiosystems, Huntsville, AL, USA). Proliferation andmigration were analysed by counting cumulative cell numbers and byperforming transwell assays in the absence or presence of EGF.Results: All AKT isoforms were detected in the different trophoblast cellmodels using real-time PCR and Western blotting. Transcript and proteinlevels of AKT did not largely vary between the different trophoblastcultures. Western blot analyses revealed that the extent of knock-downwas between 80% and 90% for the different AKT isoforms. Compared tonon-silencing controls, knock-down of AKT1, AKT2, and AKT3 did notaffect trophoblast proliferation. Basal migration was only diminished inAKT3 knock-down cells, whereas EGF-dependent migration was stronglyreduced in both AKT1 and AKT3 gene-silenced pools. AKT2 knock-downhad only subtle effects of EGF-dependent migration.Conclusion: None of the different AKT isoforms is involved in trophoblastcell proliferation. Basal migration may largely depend on AKT3. AKT1 andAKT3 seem to play redundant roles in EGF-dependent trophoblastmigration.S.S. and P.H are supported by grant P-17894-B14 of the Austrian ScienceFunds, K. Biadasiewicz is supported by grant Nr. 12487 of the AustrianNational Bank, Austria.Keywords: trophoblast, invasion, AKT, signaling

[P05.13].IDENTIFICATION OF VEGFR-2 AS A NOVEL DECORIN BINDING RECEPTORON THE HUMAN EXTRAVILLOUS TROPHOBLAST CONTROLLINGACQUISITION OF AN ENDOVASCULAR PHENOTYPE

G Khan*1,2, N Lala1, G Gannareddy1, RN Bhattacharjee1, PK Lala1. 1Universityof Western Ontario, Canada, 2Defense Institute of Physiology and AlliedSciences, New Delhi, India

Introduction: The human placenta is an invasive structure in which a cellpopulation known as the extravillous trophoblast (EVT) migrates out ofchorionic villi and invades the uterine endometrium and its arteries,adopting an endovascular phenotype. Poor uterine arterial invasion andremodelling by EVT cells is associated with IUGR in the fetus andpreeclampsia in the mother. We have identified two decidua-derivednegative regulators of EVT cell proliferation, migration and invasiveness:TGF-beta, and a TGF-beta binding small leucine rich proteoglycan decorin(DCN) co-localised with TGF-beta in the decidual ECM (Xu, et al. BiolReprod 67, 681-89,2002). DCN actions on EVT cells were differentiallymediated by multiple tyrosine kinase receptors EGF-R, IGFR-1 and VEGFR-2(Iacob, et al. Endocrinol 149,6187-97,2008).Objectives: Since DCN binding to VEGFR-2 has never been reported beforein any cell type, we directly tested this binding and the identity of VEGFR-2binding sites of DCN protein in our human first trimester EVT cell line HTR-8/SVneo, and further examined whether this binding could retard VEGF-induced acquisition of an endovascular phenotype.Methods and Results: EVT cell lysate proteins were subjected to far-western blots by using purified DCN as ‘‘bait’’ and VEGFR-2 as ‘‘prey’’proteins. DCN binding to EVT cell proteins was detected with DCN antibodyand re-probed with VEGFR-2 antibody, showing DCN-bound VEGF-R2.Pure VEGFR-2 served as positive control. Similar proof was obtained by co-immunoprecipitation of DCN and VEGFR-2 in EVT cell lysate proteins,probed with DCN antibody. Certain DCN peptides that preferentiallyblocked DCN-VEGR-2 binding in EVT cell proteins also blocked EVTproliferation, indicative of possible VEGFR-2 binding sites of DCN. In a cellfree system, using surface plasma resonance spectroscopy, we approxi-mated the affinity of binding between pure DCN and pure VEGFR-2,providing a dissociation constant (Kd) of 73 mM. Finally, EVT cells whenplated on growth factor reduced matrigel formed sparse endothelial-liketubes (acquisition of endovascular phenotype), which was stimulated inthe presence of VEGF121. This VEGF-induced stimulation was inhibited byDCN pre-treatment in a dose-dependant manner.Discussion: That DCN binds to VEGFR-2 is a novel finding for any cell type.DCN-VEGFR-2 interactions in controlling acquisition of an endovascularphenotype by EVT cells may suggest that DCN over expression or activitymay contribute to the development of preeclampsia. We are currentlytesting this possibility. (Supported by a CIHR grant to PKL)Keywords: Extravillous trophoblast, Decorin, VEGFR-2, Preeclampsia


[P05.14].ENDOGLIN IN HUMAN EXTRAVILLOUS TROPHOBLAST INDUCED BYTGF-BETA

Y Mano*, T Kotani, S Sumigama, H Hayakawa, K Shibata, F Kikkawa. NagoyaUniversity Graduate School of Medicine, Japan

Endoglin (CD105) is a co-receptor for transforming growth factor-beta(TGF-beta). Endoglin is known to be expressed in syncytiotrophoblasts andits soluble form increases in preeclamptic serum. On the other side, severalstudies showed that endoglin was involved in cancer invasion. However,the role of endoglin in human trophoblast function remains unknown. Theaim of this study was to investigate the expression of endoglin in evtra-villous trophoblast (EVT), which poses invasive phenotype. We showedthat endoglin was expressed on interstitial and endovascular EVT inhuman first-trimester implantation site by immunohisctochemistry. Then,we investigated what factors would control the expression of endoglin.Semi-quantitative RT-PCR and Western blotting revealed that theexpression of endoglin was increased under hypoxia (1%O2) condition.Both TGF-beta1 and TGF-beta3 (10ng/ml) also significantly inducedendoglin in human EVT cell line HTR-8/SVneo cells. We gained primarycultured EVT from first-trimester human villous explants culture. Inprimary cultured EVT, the similar results were observed. These data sug-gested that endoglin was expressed in EVT, and the expression is regulatedby hypoxia, TGF-beta1 and TGF-beta3. Now we are going to investigate therole of endoglin in EVT to introduce siRNA of endoglin into HTR-8/SVneo.

Keywords: endoglin, extravillous trophoblast, transforming growthfactor-beta, hypoxia

[P05.15].THE ROLE OF AUTOPHAGY ON THE INVASION OF EXTRAVILLOUSTROPHOBLAST

Akitoshi Nakashima*1, Mikiko Tatematsu1, Tamotsu Yoshimori2, ShigeruSaito1. 1University of Toyama, Japan, 2University of Osaka, Japan

Objectives: Extravillous trophoblast (EVTs) suffer from severe environ-ment, such as hypoxia and low nutrition, during early pregnancy. Gener-ally, hypoxia reduces cell viability, but EVTs maintain the invasiveness forsuccessful pregnancy. Autophagy (AtP) is a bulk degradation system forpromoting cell survival under nutrient depletion. In this study, we showedthe specific role of autophagy on EVTs-invasion under hypoxia by usingautophagy-defect cell line.Methods: EVT cell line, HTR-8/SVneo cells (HTR8) were incubated inDMEM with CoCl2 250mM (2%O2). We constructed AtP-defect cell line,HTR-4B, which is stably transfected with Atg4B dominant negative mutantby retrovirus vector. Using this cell line, intracellular ATP quantificationwas examined by luciferase driven bioluminescence. MMPs-mRNA levelswere analyzed by real time RT-PCR.Results: We provided the three major findings in IFPA meeting 2008.1) Autophagy occurred in EVT primary cells and HTR-8 under hypoxia.2) 3-MA, AtP-specific inhibitor, significantly reduced the numbersof invading cells under hypoxia but not normoxia. 3) AtP was observedin EVT cells in human early pregnant specimen. In this study, weestimated the number of invaded cells between HTR8-4B, and HTR8-cont.Hypoxia significantly decreased the numbers of invaded cells by 81% inHTR8-4B, compared with normoxia, whereas hypoxia increased that ofcells by 153% in HTR8-cont. No significant differences were detectedbetween HTR8-4B and HTR8–cont in the growth rate and the cell deathrate under hypoxia. Intracellular ATP levels were significantly decreased inHTR8-4B, but not in HTR8-cont under hypoxia. Furthermore, MMP2,MMP9 and uPA levels were also significantly decreased in HTR8-4Bcompared with HTR8-cont.Conclusions: These findings suggested that autophagy produced energy toEVT cells, which were invading to decidua under hypoxia. On this process,autophagy machinery may interact with producing MMPs on invadedEVT cells.Keywords: Autophagy, Hypoxia, ATP, MMPs

[P05.16].EFFECTS OF ADIPONECTIN ON DIFFERENTIATION, INVASION ANDMIGRATION OF HUMAN TROPHOBLASTIC CELLS

A Onogi*, K Naruse, H Shigetomi, Y Yoshizawa, T Noguchi, T Sado.Dept. of Obstetrics & Gynecology, Nara Medical University, Japan

Introduction: Adiponectin (Adn) is an adipocyte-derived cytokine leadsinsulin sensitivity and anti-inflammatory action through TNF-a-suppres-sion. We reported a paradoxical increase of Adn in preeclampsia (PE), butthe manner of Adn effects on trophoblasts remains unclear. In this study,we cultured trophoblasts separated from human placenta and tropho-blastic cell lines with different concentrations of Adn and evaluated theeffects of Adn in several methods.Methods: Human term placentas were taken at selective cesarean sectionwith informed consent. Separated trophoblasts by Percoll gradient wereharvested on fibronectin-coated plate overnight with or without Adnaddition. Cytotrophoblasts(CTBs) were separated from early terminatedpregnancy sample with similar method and cultured on MatrigelR over-night. Cytokines / proteinases / inhibitors in supernatants were measuredwith Multiple Cytokine Assay after confirmation of cell viability using MTTassay. Immunocytochemistry for cytokeratin and HLA-G were performedon early CTBs to assess the migration/differentiation to invasive pheno-type. Adn were also added in several concentrations at culture oftrophoblast cancer cell line JEG-3, JAR and BeWo, and invasion assay andwound healing assay were performed.Results: Effects of high concentration Adn culture of term trophoblastswere not significant on cytokines / proteases / inhibitors secretion and cellviability. Addition of Adn in primary CTBs culture from early pregnancyincreased connection and differentiation of the cells in lower concentra-tions, but inhibited them in higher concentrations. Ability of invasion afterAdn addition were varied between cell lines, but significant increase wasshown in JAR after 10mg/ml Adn addition (p<0.05). Migration was signif-icantly decreased in BeWo in 100ng/ml Adn (p<0.05), but not significant inother higher or lower concentrations.Conclusion: Differentiation, migration and invasion of cytotrophoblastfrom early pregnancy and cancer cell line were strongly effected by Adn.Rather than the possible effects of Adn on placenta in PE, these might bebasic evidences to explain the high rate of recurrent miscarriage or fetalgrowth restriction in hypoadiponectinaemia, like diabetes mellituscomplicated pregnancy.Keywords: Adiponectin, trophoblast invasion, trophoblast migration,diabetes mellitus


[P05.17].INTERLEUKIN-11 INHIBITS HUMAN TROPHOBLAST INVASION VIASTAT-3 INDICATING AN IMPORTANT ROLE DURING PLACENTALDEVELOPMENT

P Paiva*1,2, L Salamonsen1,2, U Manuelpillai2, E Dimitriadis1,2. 1PrinceHenry’s Institute of Medical Research, Australia, 2Department of Obstetricsand Gynaecology, Monash University, Australia

Successful pregnancy depends on the precise regulation of extravilloustrophoblast (EVT) invasion into the uterine decidua, primarily by decidua-derived factors. In humans, during early pregnancy, interleukin (IL)-11 ismaximally expressed in the decidua1, with its receptor, IL-11-receptoralpha (Ra) also identified on invasive EVT in vivo2. While a role for IL-11 inEVT migration has been established2, whether it also plays a role inregulating EVT invasion is unknown. We investigated whether IL-11influences human EVT invasion and the signalling pathways and under-lying mechanisms involved using the HTR-8/SVneo immortalized EVT cell-line and primary EVT as models for EVT. The effect of IL-11 on tyrosinephosphorylation (p) of signal transducer and activator of transcription(STAT)-3 was determined by Western Blot. EVT invasion was assessedusing in vitro Matrigel invasion assays. To elucidate the mechanisms bywhich IL-11 may influence EVT invasion, matrix metalloproteinase (MMP)and urokinase plasminogen activator (uPA) activity were assessed bygelatin and plasminogen zymography / uPA activity assay respectively.Tissue inhibitor of MMPs (TIMPs)-1 and -2, plasminogen activator inhib-itor (PAI)-1 and -2 and uPA receptor (uPAR) were assessed by ELISAwhereas TIMP-3 was assessed by Western Blot. EVT adhesive propertiesand integrin expression were assessed by in vitro adhesion assays. IL-11(100 ng/ml) significantly inhibited invasion of EVT cells by 40-60%(p<0.001). This effect was abolished by inhibitors of STAT-3 but not ofmitogen-activated protein kinase pathways. IL-11 (100 ng/ml) had noeffect on MMP-2 and -9, TIMP 1-3, uPA, uPAR, PAI-1 and -2 in EVT condi-tioned media and / or cell lysates. IL-11 (100 ng/ml) also did not regulateEVT cell adhesion or integrin expression. These data demonstrate that IL-11 inhibits human EVT invasion via STAT-3 indicating an important role forIL-11 in the decidual restraint of EVT invasion during normal pregnancy.References(1) Dimitriadis et al. (2003) Reprod Biol Endocrinol. 1, 34-38.(2) Paiva et al. (2007) Endocrinol. 148, 5566-72.Keywords: Interleukin 11, STAT3, Trophoblast

[P05.18].EXPRESSION OF PREGNANCY RELATED SERINE PROTEASE HtrA3 ISTIGHTLY UPREGULATED DURING HUMAN STROMAL CELLDECIDUALIZATION

H Singh*, L Salamonsen, G Nie. Prince Henry’s Institute of MedicalResearch, Melbourne, Australia

Background: Adequate invasion of the trophoblast cells is necessary forimplantation and placentation. Expression of serine protease HtrA3 ishighly upregulated in the decidualizing stromal cells in late secretoryphase of the menstrual cycle and throughout pregnancy. It is highlyexpressed in the 1st trimester in most trophoblast cell types, but not in theinvading interstitial trophoblast. Unlike cancer cells in which HtrA3 isdown-regulated, trophoblast generally exhibit controlled invasion. Thecurrent study investigated expression of HtrA3 during decidualization ofhuman endometrial stromal cells (HESC) in vitro and its function.Methods: Stromal cells were isolated from normal human endometrialbiopsies (n¼3) and decidualized in vitro with estrogen, progesterone andcyclic-AMP. HtrA3 expression at various time points (0 - 96h) in decid-ualized and non-decidualized HESC was assessed by quantitative RT-PCR.Indirect immunofluorescence and western was performed to determinethe cellular localisation and protein expression of HtrA3.Results: Both HtrA3 mRNA and protein expression was significantlyincreased in decidualized stromal cells compared to controls. HtrA3mRNA expression for both isoforms (long and short) was significantlyincreased within the first 48h of decidualization. Approximately 25-40fold increase was observed within initial 24h of decidualization for thelong and short form respectively reaching maximum levels at 48h. Thelevel of decidual HtrA3 mRNA gradually decreased with further decidu-alization, but expression level remained significantly higher thancontrols. Homogeneous pattern and increase in intensity of HtrA3 stain-ing was observed in decidualized stromal cells at 96h in comparison tonon-decidualized cells.Conclusions: HtrA3 is tightly regulated during decidualization of HESC invitro. It might be possible that the significant initial increase of HtrA3expression has a role in promoting decidualization. Function of HtrA3 introphoblast invasion and its regulation in normal and complicated preg-nancies will be explored.Keywords: HtrA3, Decidualization, Trophoblast, Invasion


[P05.21].EXPRESSION OF N-ACETYLGLUCOSAMINYLTRANSFERASE IVa INEXTRAVILLOUS TROPHOBLAST AND GESTATIONAL TROPHOBLASTICDISEASE

E Yamamoto*, K Niimi, K Hayashi, T Kotani, K Ino, F Kikkawa. NagoyaUniversity, Japan

Objectives: Hyperglycosylated hCG (hCG-H) is a glycoprotein variant ofthe hormone hCG and reported to be detected in serum and urine of earlypregnant women, invasive mole and choriocarcinoma, but not of molarpatients. N-acetylglucosaminyltransferase IVa (GnT-IVa) is one of glyco-syltransferases to attach abnormal biantennary N-linked sugar chains tohCG. Our aim is to clarify the expression of GnT-IVa in gestationaltrophoblastic disease and extravillous trophoblasts (EVTs) in humanplacenta.Methods: Western blot and RT-PCR were performed for expression levelsof GnT-IVa protein and mRNA in choriocarcinoma cell lines (Jar, JEG-3,Bewo, CC1, CC3, CC4 and CC6), EVT cell line (HTR-8/SVneo), variousgestational placenta and hydatidiform mole tissues. Expression andlocalization of GnT-IVa were analyzed by immunohistochemistry inchoriocarcinoma, invasive mole, hydatidiform mole and human placenta.Results: GnT-IVa was expressed in all choriocarcinoma cell lines, especiallystrongly in Jar, JEG-3, CC4, and CC6, and weakly in HTR-8/SVneo. However,GnT-IVa expression was not detected in various gestational placenta andmolar tissues by RT-PCR and Western blot. Immunohistochemical studyrevealed that GnT-IVa was strongly expressed in choriocarcinoma andinvasive mole, but not in hydatidiform mole. In human placenta, GnT-IVastaining was detected in EVT cells in the first trimester, and it was negativeall trophoblasts of the second and third trimester placentas.Conclusion: GnT-IVa was expressed specifically in choriocarcinoma,invasive mole and EVT in early gestation. These results suggested thatGnT-IVa may be involved in EVT invasion as well as malignant potential ofgestational trophoblastic disease.Keywords: GnT-IVa, EVT, Hyperglycosylated hCG, hCG

[P06.01].MYOFERLIN AND DYSFERLIN ARE DISPENSABLE FOR CELL-CELL FUSIONIN BeWo

J. M. Robinson*, W. E. Ackerman, D. D. Vandre. The Ohio State University,United States

Introduction: Cell-cell fusion is fundamental for the formation of multi-nucleated syncytia, which arise normally during the genesis of skeletalmuscle and placental syncytiotrophoblast (STB). It has recently beenshown that targeted disruption of the myoferlin (MYOF) gene in miceimpairs myoblast fusion during skeletal muscle syncytialization. Giventhat MYOF and the closely related protein, dysferlin (DYSF), are expressedin trophoblast, we speculated that MYOF might serve an analogous func-tion during cytrophoblast cell fusion.Methods: Lentivirus-based shRNA constructs were used to knock downthe expression of MYOF and DYSF in BeWo cells. Both native and knock-down cells lines were assessed for the ability to undergo forskolin-inducedfusion.Results: MYOF, but not DYSF, was expressed in mononuclear BeWo cells.Following treatment with 20 mM forskolin, DYSF expression increasedprogressively from 24 to 72 h, while MYOF expression remained constant.Three separate shRNA constructs were used to generate stable BeWo celllines exhibiting at least 80% knock down of MYOF at the protein level. Inparallel, four shRNA constructs were used to knock down DYSF expressionto a similar degree. All three of the MYOF knock-down lines (including onein which MYOF was undetectable by immunoblotting) were found toundergo forskolin-induced cell fusion to a similar degree as native BeWo.In addition, the MYOF knock-down lines retained the ability to upregulateDYSF in response to fusion. The DYSF knock-down lines exhibited MYOFexpression typical of native cells and, in the absence of DYSF upregulation,underwent normal fusion in response to forskolin.Discusson: These results suggest that neither MYOF nor DYSF are requiredfor forskolin-mediated intercellular fusion in BeWo. Importantly, thisestablishes that these cell lines can be used as a novel system in which toaddress the function of MYOF and DYSF in plasma membrane repair usingfused structures resembling STB.Keywords: Dysferlin, Myoferlin, BeWo

[P06.02].PLACENTATION MODEL IN PURANE (THRICHOMYS APEREOIDES LUND,1839).

C Amrosio*. University of Sao Paolo, Brazil

Thrichomys apereoides, a hystricognath rodent species belonging to thefamily Echimyidae, is characterized by high agility, a mainly vegetarianlifestyle, activities concentrated at dawn and a habitat in rocky areas withdense vegetation. We firstly characterize its type of placentation and theevolution of placental features. The investigated material includes fourplacentas at mid gestation, processed and analyzed by standard macro-scopy and light microscopy. The placenta possesses a disc shape with fewlobules. Lobules are clearly delimited by interlobules, with few maternallacunae. In both regions we found predominantly cytotrophoblast, but alsosyncytiotrophoblast. Complementary studies on the ultrastructure mustbe done, however the placenta can be characterized as hemochorial bymeans of light microscopy. Centrally at the junction zone there is an areawith a relatively large volume, classified as the subplacenta. The sub-placenta is not lobulated and is characterized by a large number oftrophoblast cells and syncytiotrophoblast surrounded by mesenchyme. Aninverted vitelline placenta is presented, possessing groups of giant cellsand layers of spongiotrophoblast. The visceral portion of the yolk sac haslong villous projections and is high vascularized. The parietal portion ofthe yolk sac shows just one cell layer disposed on the placenta. Insummary, placentation in Thrichomys is very similar to other members ofSouth American and African hystricognaths, indicating a remarkable stablepattern of evolutionary transformations in the placenta. Thus, includingthe guinea pig a wide range of model species is principally useable incomparison to human placentation.


[P06.03].StarD7 A FUSOGENIC PROTEIN IN TROPHOBLASTS

S.C. Angeletti1, Q. Chen2, S.M. Pearson2, S. Genti-Raimondi1,L.W. Chamley*2. 1Department of Clinical Biochemistry – CIBICI, NationalUniversity of Cordoba, Argentina, 2Department of Obstetrics andGynaecology, University of Auckland, New Zealand

StarD7 is a novel protein of unknown function that belongs to the STARTlipid domain family and is expressed in the cytoplasm of human tropho-blast cells. It was previously found that StarD7 is able to interact withphosphatidylserine. We have previously shown that StarD7 is partiallyrelocated from the cytoplasm to the plasma membrane during in vitrocytotrophoblast differentiation into syncytiotrophoblast. This studyfurther investigates the function of StarD7 in trophoblasts.MTT-based cell proliferation assays were preformed in triplicate usingBeWo cells that were incubated with increasing concentrations ofrecombinant StarD7 protein (0, 5, 10 or 20 mg/ml). Cell viability wasdetermined by propidium iodide staining and detection of LDH in theculture supernatants. Statistical analysis was by t-test and a P value < or ¼0.05 was considered to be significant. Syncytialization of BeWo cells wasassessed by immunostaining for desmoplakin.The proliferation of BeWos was significantly inhibited by incubation withStarD7 at 5, 10 and 20 mg/ml. Incubation of BeWos with StarD7 did notcause a significant increase in cell death as measured by either propidiumiodide or LDH release at any of the concentrations we tested. Fluorescentmicroscopy suggested that there were few intracellular desmosomesbetween adjacent BeWo cells after treatment with StarD7 at 20 mg/ml.BeWo cultures treated with StarD7 at these concentrations had similarlevels of intracellular desmosomes to those found in control culturestreated with 5 mM forskolin.These results together demonstrate that exogenous Star D7 causes BeWocells to cease proliferating but does not cause their death. The reduction indesmosomes indicates loss of intracellular boundaries leading us toconclude that StarD7 can initiate/facilitate the syncytialization of BeWocells. Our results suggest that the phospholipid-binding protein StarD7may play a physiological role during the differentiation of cytotrophoblastsinto syncytiotrophoblast.Keywords: START lipid domain, Trophoblast proliferation arrest, Syncyti-alization, Cell fusion

[P06.04].CROSSTALK BETWEEN EXTRINSIC AND INTRINSIC CELL DEATHPATHWAYS THROUGH CASPASE-8 IN MATERNAL FOOD RESTRICTEDRAT PLACENTAS AT E20

L Belkacemi*, MG Ross, M Desai. Department of Obstetrics and Gynecology,David Geffen School of Medicine at UCLA and LABIOMED Research Insti-tute at Harbor-UCLA Medical Center, CA, USA, United States

Introduction: Maternal food restriction (MFR) during pregnancy leads tointrauterine growth restricted (IUGR) fetuses. Placental dysfunction isamong the leading causes of IUGR. Increased placental apoptosis has beenassociated with IUGR. Apoptosis occurs via extrinsic death receptorpathway (Fas) and/or the intrinsic pathway (mitochondria). Activation ofcaspase-8 occurs directly via Fas pathway or indirectly via mitochondrialpathway. Caspase-8 activates intrinsic BID protein to form truncated BID(tBID). Using a rat model, we have shown that MFR results in reducedplacental growth and increased apoptosis at E20. Since the Fas pathway isresponsible for placental trophoblasts turnover, we sought to determinethe role of caspase-8 in the Fas-induced apoptosis. We focused our studyon the two placental positions (proximal and mid-horn) with theextremes of nutrient/oxygen supply, and two distinct placental zones(basal, site of hormone production; and labyrinth, site of feto-maternalexchange).Methods: Pregnant rat dams were fed an ad libitum diet (AdLib) or were50% MFR beginning at E10 of gestation. At E20 rats were euthanized, andgestational sacs dissected. The placentas were separated and weighed. Sixplacentas from left mid- and proximal horns were fixed in para-formaldehyde for TUNEL and activated caspase-3 staining. The corre-sponding right mid- and proximal horn placentas were separated intobasal and labyrinth zones and analyzed for the expression of caspase-8 andtBID proteins (Western blot).Results: As compared to AdLib, MFR mid- and proximal horn placentasshowed significant reduction in weight and increased apoptosis in bothzones (basal and labyrinth). Consistent with this, MFR placentas hadincreased caspase-3 expression. Furthermore, caspase-8 and tBID proteinexpression were significantly increased in both zones in mid- and proximalplacentas. Lastly, both MFR and AdLib mid-horn placentas had significantlyupregulated tBID as compared to respective proximal placentas.Conclusion: Thus, the mechanism for MFR-induced placental apoptosisvia caspase-8 appears to be mediated primarily through the extrinsic Faspathway with potentially secondary signaling from intrinsic pathway.Keywords: Apoptosis, placenta, IUGR, caspase-8


[P06.05].MATERNAL FOOD RESTRICTION IMPACTS ON MATERNAL-FETALDEVELOPMENT AND WATER HOMEOSTASIS IN RAT PREGNANCIES

L Belkacemi*, MH Beall, Q Liu, M Desai, MG Ross. Dept of Obstetrics andGynecology, David Geffen School of Medicine at UCLA and LABIOMEDResearch Institute at Harbor-UCLA Medical Center, CA, United States

Introduction: Mammalian gestations contain significant amounts ofwater, both as a major constituent of the fetus, and as amniotic fluid. Thiswater is derived from the mother across the placenta, presumably via anosmotic mechanism. We have previously shown that maternal plasmahypertonicity reduces AF volume (AFV). Maternal food restriction (MFR)during pregnancy results in intrauterine growth restricted (IUGR) fetusesand newborns. IUGR is often associated with oligohydramnios. To assessthe etiology of oligohydramnios, we examined the impact of 50% MFR onmaternal water intake, plasma osmolality and body weight, and thesubsequent effects on fetal plasma and AF osmolality and AFV at E20 (nearterm).Methods: From E10 to E20, rats were provided either an ad libitum (AdLib)diet (N¼6) or were restricted to 50% of the food of AdLib-fed rats (MFR;N¼6). From E10 to E20, MFR and AdLib dam body weights and water intakewere recorded daily and maternal plasma osmolality quantified at E20. AtE20, rat dams were euthanized and mid- and proximal horn gestationalsacs dissected. Fetal weights were recorded. AFV was quantified as thedifference in sac weight before and after drainage. Fetal plasma and AFosmolalities were quantified by freezing point depression.Results: At E10, prior to the initiation of MFR, there was no difference inbody weight between MFR and AdLib dams. At E20, MFR dams hadsignificantly lower body weights (P<0.05), decreased water intake(P<0.05) but increased plasma osmolality as compared to AdLib dams.However, the ratio of body weight to water intake was not significantlydifferent between the two groups. Fetal plasma osmolality was signifi-cantly increased in the MFR group while MFR fetal body weight wassignificantly decreased. Furthermore, AFV was significantly decreased inboth mid- and proximal horn sacs whereas the AF osmolality wasincreased in those sacs (P<0.05).Conclusion: Although MFR leads to decreased maternal body weight andwater intake at E20, the water intake adjusted for body weight werecomparable between MFR and AdLib dams, suggesting that MFR-inducedchanges are not dependent upon maternal fluid intake. However, theincreased maternal osmolality in the MFR dams may significantly alterwater flux to the fetus, leading to fetal hyperosmolality andoligohydramniosis.Keywords: Amniotic fluid, maternal food restriction, osmolality, waterhomeostasis

[P06.07].DYNAMIC STUDIES ON CHANGES IN PLACENTAL STRUCTURE ANDBLOOD FLOW IN AN ANIMAL MODEL OF PREECLAMPSIA USING HIGHRESOLUTION MRI

G Bobek*1, T Stait-Gardner1, B Bahman1, J Preis1, W Price1, A Hennessy1,2.1University of Western Sydney, Australia, 2Heart Research Institute,Australia

Introduction: The placenta appears to be central in the aetiology ofpreeclampsia. It has been postulated that reduced placental perfusion asa result of aberrant cytotrophoblast invasion and remodelling of thematernal spiral arteries is the initiating event that leads to the widespreaddysfunction of the maternal vascular endothelium. This study presents ourinitial results into the use of magnetic resonance imaging (MRI) to directlyexamine murine placental structure and placental blood flow in normalpregnancy and in preeclampsia.Methods: Embryo placenta units from 14.5 day pregnant C57BL/6JArc micewere imaged using a Bruker Avance 11.74 Tesla wide-bore spectrometerwith micro-imaging probe capable of generating gradients of 1.5 T/m. Themeasurements were made using the FLASH (Fast Low Angle SHot) methodwith echo time 6.000 ms, repetition time 397.28 ms and 20 averages (scantime w34 minutes). The field-of-view was 23.000 mm � 23.000 mm withslice thickness 0.500 mm. A 256�256 matrix size resulted in voxeldimensions of w90 mm � 90 mm � 500 mm.Results: Our results demonstrate clear and detailed structural features ofindividual embryo placental units; resolving the amniochorionicmembrane, detailed embryonic features, umbilical cord and placentalvasculature. The high resolution images enable the selection of preciseregions of interest to facilitate blood flow measurements in the placenta.

Discussion: MRI offers a non-invasive technique to conduct dynamicstudies on changes in placental structure and blood flow in animal modelsof preeclampsia. We have demonstrated a substantial enhancement inplacental image resolution above those previously reported. Using MRItechniques including BOLD (Blood Oxygen Level Dependent) MRI and DWI(Diffusion Weighted Imaging) for quantifying the flow dispersion withinthe placenta, we will be able to dynamically follow changes in placentalperfusion and structure to investigate links between cytokine imbalance,shallow placental invasion and subsequent hypertensive response.Keywords: magnetic resonance imaging, placenta, perfusion, preeclampsia


[P06.08].NITRIC OXIDE IS INVOLVED IN THE SHIGA TOXIN MECHANISMSRESPONSIBLE FOR PREMATURE DELIVERY OF DEAD FETUSES

J Burdet*1, E Zotta1, A M Franchi2, C Ibarra1. 1Laboratorio de Fisiopatogenia,Departamento de Fisiologıa, Facultad Medicina, Universidad de BuenosAires, Argentina, 2CEFYBO-CONICET, Universidad de Buenos Aires,Argentina

Shiga toxin-producing Escherichia coli (STEC) infections could be one of thecauses of fetal morbimortality in pregnant women. The main virulencefactor of STEC is Shiga toxin type 1 or 2 (Stx1, Stx2). We have previouslyreported that intraperitoneal (i.p.) injection of culture supernatant from E.coli recombinant expressing Stx2 and containing lipopolysaccharide (LPS)in rats in the late stage of pregnancy induced premature delivery of deadfetuses.It has been reported that LPS may combine with Stx2 to facilitate vascularinjury that may lead to a pathological cascade that involves the productionof nitric oxide (NO).Objective: Our aim was to evaluate if NO is involved the effects of Stx2 onpregnancy.Materials and Methods: Pregnant rats on days 14-16 of gestation were i.p.injected with culture supernatant from recombinant E. coli containing 0.4mg/ml Stx2 and 30 ng/ml LPS.A group of rats was previously injected with aminoguanidine (AG), aninducible NO synthase (iNOS) inhibitor and the development of pretermlabor was evaluated.Western blot analyses were performed to determine the iNOS expressionin placentas from Stx2-treated and untreated rats.Results: Stx2 and LPS induced fetal resorption, placental abruption,intrauterine hemorrhage and fetal death at 1-2 days post-injection. Pre-treatment of 24 h with AG caused significant reduction of Stx2 effects onthe feto maternal unit but did not prevent the premature delivery of deadfetuses.Histological studies show no significant differences in placenta tissuesfrom rats treated with AG and Stx2 compared with those treated only withAG or with controls.Western blot assays showed a higher expression of iNOS in placentas fromStx2-treated rats than in those previously treated with AG.Conclusions: Our results suggest that NO is partially involved in themechanisms of the premature delivery of dead fetuses caused by Stx2 andLPS.Keywords: Shiga toxin, preterm labor, pregnancy, nitric oxide

[P06.10].A CASE OF A RETAINED PLACENTA PERCRETA FOLLOWED BY A VIABLEBIRTH FIVE YEARS LATER

H.C. Chihara*, Y.N. Nagai, T.W. Watanabe, T.A. Adachi, A.N. Nagai, K.T.Tsutsumi. Department of Obstetrics and Gynecology, Nagai Clinic, Japan

Placenta accreta is considered to be due to the absence of the deciduabasalis and the associated invasion of the myometrium by the placentalvilli that may reach the peritoneal covering. We report on our experiencewith a patient in whom placenta increta recurred at the site of a previousplacenta percreta, but who delivered a live baby and the uterus was saved.The patient was a 33-year-old multipara with two previous deliveries byCaesarean section. At the age of 28 years, she conceived naturally anddelivered her second child by caesarean section. However, in this pregnancyshe was found to have a placenta percreta that was attached over an area fromthe uterine body to the fundus. Since it was impossible to detach the placenta,the operation was terminated without removing it. The retained placentagradually shrank and apparently mostly disappeared during the followingyear, until only remnants of the placenta remained on the serous membrane.On magnetic resonance imaging (MRI), the myometrium at the uterinefundus was thinning or missing, while the serous membrane was preserved.After 5 years, the patient conceived naturally. The placenta was attached inthe region of the previous placenta percreta. According to the observationsmade during the Caesarean section performed at 37 weeks of gestation,the diagnosis was again placenta increta, but this time it could be sepa-rated, and the uterus could be saved.This case study illustrates the reparative capacity of the endometrium andshows that it is not always necessary to perform a hysterectomy inuncomplicated abnormal implantations of the placenta. It also demon-strates the time course of resorption of retained placenta percreta tissues.We will also report on the peri-operative findings from the Caesareansection in the third pregnancy, including ultrasound images, MRI resultsand hysteroscopic appearance.Keywords: placenta accreta, placenta percreta, magnetic resonanceimaging, hysteroscope


[P06.13].ALTERED INSULIN RECEPTOR SPLICING RESULTS IN DIFFERENTIALINSULIN SIGNALLING IN GDM

U. Hiden1, E. Glitzner1, L. Lassance1, I. Cetin2, U. Lang1, G. Desoye*1. 1MedicalUniversity Graz, Austria, 2University of Milano, Italy

Objectives: The human insulin receptor (IR) exists in two isoforms thatdiffer by inclusion/exclusion of exon 11, hence IR11+ and IR11-. Exonskipping results in different signalling efficiency and ligand affinity. Dia-betes alters IR splicing in muscle, liver and adipose tissue. Here, wehypothesized altered insulin receptor splicing in the placenta resultingfrom the diabetic environment in GDM, which may further alter insulinsignalling.Methods: IR splicing was determined in normal (n¼28) and GDM (n¼17)placentas using RT-PCR. The mRNA expression of IR splicing factors wasmeasured to identify factors involved in splicing alterations. Cytokeratin-7and vWF were measured to exclude that changes in cellular compositionaccount for altered IR splicing patterns. Insulin signalling differences inIR11+ vs. IR11- was determined by immunoblotting of Erk1/2 and PKBactivation in mouse NIH3T3 cells over-expressing human IR11+ or IR11-.Isolated placental endothelial cells were treated in vitro by hyper-insulinemia and hyperglycaemia to identify possible factors altering IRsplicing in vivo.Results: IR11+ over-expressing cells activated the Erk1/2 pathway 5-foldstronger than the PKB pathway. In contrast, IR11- over-expressing cellsinduced PBK signalling 3-fold stronger than Erk1/2 signalling. In GDM, theamount of IR11- was reduced by 29% (p¼0.03) whereas IR11+ remainedunchanged. Changes in prevalence of IR isoforms did not result fromaltered placental cellular composition as no change was found in theproportion of CK7 to vWF. In isolated placental endothelial cells, IR11-proportion was reduced by 15% after treatment with glucose (12mM) plusinsulin (1nM).Conclusions: Reduced placental expression of IR11- vs. IR11+ in GDM givesrise to altered insulin signalling and effects. In vitro experiments indicatethat altered insulin receptor isoform expression may result from hyper-glycaemia and hyperinsulinemia in the fetal circulation that affect IRsplicing in endothelial cells. As a consequence these may show reducedPKB mediated insulin effects.(Jubilee Fund grant no: 10896, 12601, 13307)Keywords: insulin, signalling, gestational diabetes

[P06.14].AUTOMATED CELL-DETECTION TECHNOLOGIES FOR SCIENCE ANDDIAGNOSTICS

A. Heindl1, R. Ecker2, G. Bises1, T. Thalhammer1, I. Ellinger*1, A. Seewald3.1Medical University Vienna, Austria, 2TissueGnostics, Austria, 3SeewaldSolutions, Austria

Introduction: As molecular cell phenotypes in cell-cohorts and tissues,also referred to as cytomes, result from genotype and environmentalexposure, there exists phenotype-heterogeneity in healthy tissues that ispronounced in case of disease. These different phenotypes containinformation about the present disease status (diagnosis) as well as itstherapy dependent future development (prediction). Consequently, thereis growing interest in basic medical research and diagnostics for multi-molecular analysis of cytome heterogeneity in combination withexhaustive bioinformatic knowledge extraction (cytomics). Cytomics-technologies are often microscope-based (slide based cytometry) usingautomated in-situ identification of cells and quantification of associatedmarker molecules. However, automated in-situ identification of specialcellular shapes (e.g. multi-nucleated cells, cells without nucleus, andrecognition of sub-cellular compartments) by means of high-contentimage analysis has not been established properly so far. Our project aimsto find a new approach for holistic pattern-recognition based on human-like interpretations.Methods: Classical digital image-processing and pattern recognitionapproaches combined with machine-learning techniques are used toaccess also implicit expert knowledge. Cell recognition will be improved byincluding multiple cellular parameters like nuclei, generic markers, cell-type specific markers in the analysis in comparison to current state-of-the-art systems.Results: This versatile system is developed and primarily applied onsections of tissues that are not able to be analyzed with the current state-of-the-art technology. The unique functionality of this approach is exem-plified by demonstrating placental tissue with its multinuclear syncytio-trophoblast and endothelial cells lacking nuclei due to the preparationprocedure. A first application of the novel automated cell-detection anal-ysis is done in a biomedical pilot project on the characterization of the IgGtransport pathway in placental chorionic tissue.Discussion: This study aims on improvement of automated in-situ iden-tification of various placental cell-types in order to allow application ofcytomics state-of-the-art technologies in placental basic research but alsodiagnosis.Keywords: Slide-based cytometry, cytomics, automated microscopy, IgGtransport


[P06.17].PLACENTAL BARRIER IN NECROMYS LASIURUS AND ORYZOMYS SP.(CRICETIDAE: SIGMODONTINAE)

PO Favaron1, AM Carter2, CE Ambrosio1, AC Morini1, ALR Franciolli1,MA Miglino*1. 1Sao Paulo University, Brazil, 2University of SouthernDenmark, Denmark, 3Universidade Federal Rural do Semi-Arido, Brazil,4Humboldt-University Berlin, Germany

The family Cricetidae has 681 recognized species. It includes Sigmo-dontinae, an important subfamily of Neotropical rodents, for which thereis little information on placenta and placentation. This study focuses on thecharacteristics of the placental barrier in Necromys lasiurus and Oryzomyssp. Four placentae from each species were obtained from Collection ofZoology Museum of Sao Paulo University, Brazil. Labyrinth tissues werefixed in 2.5% glutaraldehyde and prepared for TEM standard procedures.The placental barrier in these species consists of three trophoblast layers(TI, TII and TIII) and the fetal endothelial cell. TI is closest to maternal bloodspaces; it consisted of a thin membrane, often discontinuous and hadrelationship with TII. Due to the strong contact between TI and maternalblood spaces, maternal blood cells were often observed to be surroundedby TI. The middle layer TII or middle layer showed a villous appearancewith projections that on account of the discontinuous structure of TI insome places were in close contact with both TII and maternal blood. Cellsof TII were dispersed in the extracellular matrix, had a single nucleus andlittle organized nucleolus with areas of condensed chromatin dispersedthrough the nucleus. TIII had a very strong relationship with the fetalcapillary endothelium basement membrane as junctions were observedjunctions in the area of apposition of these membranes. TIII was connectedto TII by close and intermediate junctions, and desmosomes. The labyrinthis the region of most contact between maternal and fetal circulations. ThusN. lasiurus and Oryzomys sp. showed three trophoblast layers separatingthe two blood streams, so the placenta is classified as haemotrichorialsubtype as in other Cricetid species described by Carpenter (1972 and1975), King and Hastings (1977), Takata (1997), and Limongi and Ferro(2003).Keywords: placentation, rodents, haemotrichorial

[P06.18].HIGH D-GLUCOSE UP-REGULATES hCAT-1 EXPRESSION AND ACTIVITYBY INCREASING SLC7A1 EXPRESSION IN HUMAN UMBILICAL VEINENDOTHELIUM

Marcelo Gonzalez*1,2, Paola Casanello1,2, Luis Sobrevia1,2. 1Pontificia Uni-versidad Catolica de Chile, Chile, 2Cellular and Molecular PhysiologyLaboratory, Chile

L-Arginine is taken up by human umbilical vein endothelial cells (HUVEC)via the cationic amino acid transporter 1 (hCAT-1) isoform encoded bySLC7A1 gene. High D-glucose increases L-arginine transport, but mecha-nisms regulating hCAT-1 expression are unknown.Methods: Primary cultures of HUVEC (passage 2) were cultured understandard conditions. hCAT1-mediated L-arginine transport (0-1000 mM L-arginine, 2 mCi/ml L-[3H]arginine, 37�C), hCAT1 protein abundance andmRNA copies were determined.Results: HUVEC exposed to D-glucose (5-25 mM, 0-24 hours) exhibitincreased L-arginine (100 mM) uptake at 8 and 24 hours of incubation,associated with higher maximal velocity (Vmax) and capacity (Vmax/Km) ofL-arginine transport (EC50¼ 15� 0.2 mM). High D-glucose increases hCAT-1 mRNA expression (4 and 24 hours) and protein abundance (8 and 24hours), an effect blocked by actinomicyn D. SLC7A1 promoter transcrip-tional activity was higher in high D-glucose (24 hours) as well as the Sp1nuclear abundance and binding to SLC7A1 promoter.Conclusion: D-Glucose increases L-arginine transport by increasing hCAT-1 expression, likely due to increased Sp1 binding to SLC7A1 promoter inHUVEC.Supported by FONDECYT 1070865/1080534, CONICYT 23070213. M.Gonzalez holds a CONICYT-PhD fellowship.Keywords: L-arginine transport, High glucose, hCAT-1, endothelium


[P06.19].IGF-2 RECEPTOR SIGNALLING AND TROPHOBLAST CELL SURVIVAL INTERM PLACENTAL EXPLANTS

LK Harris*, JD Aplin, PN Baker, IP Crocker, M Westwood. University ofManchester, United Kingdom

Introduction: Insulin-like growth factor-II (IGF-II) enhances proliferationand survival of cytotrophoblast by signalling through the IGF-1 receptor.Classically, the IGF-2 receptor (IGF-2R) is thought to serve as a clearancerather than a signalling receptor, by trafficking excess IGF-II for lysosomaldegradation. We hypothesized that knockdown of IGF-2R in the syncy-tiotrophoblast of term placental explants would enhance cytotrophoblastresponsiveness to IGF-II.Methods: Term placental explants were transfected with siRNA specific forIGF-2R or a control sequence; knockdown was validated by RT-qPCR andimmunohistochemistry. 48h post-transfection, explants were transferredto serum-free culture medium and stimulated with IGF-I, IGF-II orLeu27IGF-II, which only binds IGF-2R. After a further 24h, proliferation andapoptosis were assessed by immunostaining for Ki67 and M30,respectively.Results: siRNA decreased IGF-2R mRNA by 96.5% after 48h, and reducedprotein expression in syncytiotrophoblast. In control explants, all IGFtreatments increased the number of Ki67-positive cytotrophoblasts(P<0.001; 2-way ANOVA); however, Leu27IGF-II did not enhance prolif-eration after IGF-2R knockdown. Similarly, all IGF treatments decreasedthe number of M30-positive cells in control explants (P<0.001), butLeu27IGF-II did not decrease apoptosis after IGF-2R knockdown. As pre-dicted, IGF-II-mediated rescue of cytotrophoblast apoptosis was increasedafter IGF-2R knockdown, relative to IGF-II-treated control explants.However, IGF-2 had no additional effect on cytotrophoblast proliferation inexplants with reduced IGF-2R.Discussion: The absence of IGF-2R may increase the availability of IGF-2for signalling through IGF-1R, resulting in enhanced cell survival.Mitogenic and pro-survival effects of Leu27IGF-II were only observed inthe presence of IGF-2R, suggesting that this ligand may displace IGF-2from IGF-R2 and/or signal through IGF-2R; further work is required todelineate the mechanisms involved. As increased cytotrophoblastapoptosis is observed in pregnancies complicated by fetal growthrestriction, development of strategies to decrease IGF-2R expression inthe syncytiotrophoblast may provide a therapeutic avenue for treatingthis condition.Keywords: cytotrophoblast proliferation, cytotrophoblast apoptosis,IGF-2, FGR

[P06.20].CASPASE DEPENDENT REMODELLING OF CYTOSKELETAL ALPHA-FODRIN IN FUSING TROPHOBLASTS AND BeWo CELLS

Martin Gauster*, Monika Siwetz, Kristina Orendi, Gerit Moser, GernotDesoye, Berthold Huppertz. Medical University Graz, Austria

Introduction: Fusion of cytotrophoblasts with the syncytiotrophoblast isan essential step in differentiation of the human villous trophoblast.While knowledge about potential fusogenic factors deepened in therecent past, details of membrane cytoskeleton remodelling duringtrophoblast fusion are not yet identified. This study focussed on remod-elling of submembranous spectrin-like a-fodrin during intertrophoblasticfusion.Methods: Primary term trophoblasts and forskolin challenged BeWo cellswere subjected to quantitative real-time RT-PCR, immunofluorescence andimmunoblotting analyses to elucidate expression, localization and frag-mentation of a-fodrin. Calpain inhibitors (calpeptin and calpain inhibitorIII), inhibitors of caspases 3, 8 and 9 (DEVD, IETD and LEHD) and a generalcaspase inhibitor were applied to identify involved proteases in a-fodrinfragmentation.Results and Discussion: Experiments with primary trophoblasts andforskolin challenged BeWo cells revealed a biphasic strategy of the cells toachieve reorganization of a-fodrin during fusion.One strategy was down-regulation of a-fodrin mRNA, which was observedearly in fusing trophoblasts and BeWo cells. The second strategy wasproteolytic fragmentation of already existing full-length a-fodrin, whichwas cleaved into 120 and 150kDa fragments. Application of functionalcalpain inhibitors did not block a-fodrin fragmentation, suggesting that a-fodrin remodelling is a calpain independent process during trophoblastfusion. Inhibitors of caspases 3, 8 and 9, however, attenuated generation ofthe 120kDa fragment, indicating that all three caspases participated in thecleavage process. In fusing BeWo cells activation of caspases 3, 8 and 9 wasdetected. The fact that a general caspase inhibitor completely blockedfragmentation (120 and 150kDa fragments), argues for an exclusive role ofcaspases in a-fodrin remodelling in trophoblasts. Finally, immunofluo-rescence double staining of human first trimester placenta revealed co-localization of active caspase 8 with a-fodrin positive vesicles in fusingvillous cytotrophoblasts.These results suggest that a bundle of caspases mediate remodelling ofsubmembranous a-fodrin during trophoblast fusion.Keywords: villous trophoblast, syncytial fusion, caspase, calpain


[P06.22].MMP-2 EXPRESSION IN PLACENTAE AT HIGH ALTITUDE PREGNANCY

F Lyall*1, L Marks1, S Zamudio2. 1University of Glasgow, United Kingdom,2New Jersey Medical School, United Kingdom

Introduction: Metalloproteinases (MMPs) have been implicated introphoblast invasion and placental vascular remodelling. The expression ofsome MMPs may be regulated, at least in part, by changes in oxygentension. Pregnancies at high altitude are exposed to chronic hypoxia andwe have previously shown that villous endothelial MMP-9 expression isreduced in high altitude pregnancies compared with moderate altitude orsea level pregnancies1.Methods and Hypothesis: We hypothesised that MMP-2 expressionwould also be altered at high altitude. Placentae were studied fromuncomplicated pregnancies at sea level (n¼5), moderate altitude, 1600m(n¼6) and high altitude 3100m (n¼9). Immunohistochemistry was used todetermine MMP-2 expression on villi. Immunostaining was quantified byan investigator blinded to the tissue identity and an arbritary scale of 0-4.The data was tested using the Shapiro-Wilk analysis and was not normallydistributed hence non-parametric analysis was used.Results: MMP-2 was expressed in trophoblast, endothelium, muscle andstroma at all altitudes. In contrast to MMP-9 no difference in immunos-taining was found between the two groups in any of the cell types. Therewas a trend for reduced endothelial staining but this did not reachstatistical significance.Conclusions: In contrast to MMP-9 we have found no evidence to supportthe hypothesis that MMP-2 is abnormally expressed in the placenta, atterm, in uncomplicated pregnancies at high altitude. Whether enzymeactivity is altered requires further investigation.1Marks L, Zamudio S & Lyall F (2002) MMP-9 expression is abnormal inplacentae at high altitude: A link to chronic hypoxia? Hypertens Preg 21(suppl 1) 111Keywords: oxygen, high altitude, MMP, immunohistochemistry

[P06.23].ENDOMETRIAL BEHAVIOUR OF COATI (NASUA NASUA)

JC Morini Junior*1, PO Favaron1, AC Morini1, ALR Franciolli1, MA Miglino1, CEAmbrosio2. 1School of Veterinary Research and Animal Science, Brazil,2Faculty of Animal Science and Food Engineering, Brazil

Two species of coatis, Nasua narica and Nasua nasua lives from Arizona toArgentina. Coati (Nasua narica) and racoon placentation have beenobserved, but less information is available about Nasua nasua. Theseaspects are important to be studied for comparative placentation espe-cially with another carnivore species. This study focuses on the charac-teristics of the uterus that was supposedly pregnant obtained fromUNIFEOB trial center, Sao Paulo, Brazil. The possible pregnancy was donebecause of a less increase of one of the horns and also the presence ofcorpus luteum at the ovarium of the same side. Tissues were fixed in 10%formaldehyde and prepared for light microscopy and immunohisto-chemistry by standard procedures. The bicornual uterus was composedby perimetrium, myometrium, and endometrium. At the myometriumwere found a large number of vessels strongly stained by vimentin. Twodistinct arrangement of muscle cells were observed at this layer, onecircular and other longitudinal separated by vessels. The endometriumconsists in a simple cuboidal epithelium with numerous glands with highactivity of secretion. Those glands and the endometrium layer of thelumen present cytoqueratin positive stain at its cytoplasm. As we know,cytoqueratin was expressed at the cytoskeleton of trofoblast cells, but itwas found at the lumen of endometrial gland cells, however, a disorga-nized area was found related to embryo site of implantation. The embryocannot be found, and because of it we cannot confirm the gestation, butthe high activity of the uterine glands its rearrangement more deep andthe positive stain of cytokeratin gave us evidence that the maternalenvironment was prepared for the embryo attachment as know indomestic carnivores.Keywords: Coati, Placenta, Uterus, Immunohistochemistry


[P06.24].ENDOTHELIAL PROGENITOR CELLS ARE NOT DECREASED IN MATERNALSERUM FROM PATIENTS WITH DIABETES IN PREGNANCY

DW Morrish*, J Dakour, C Chik, A Shuaib. University of Alberta, Canada

Introduction: Endothelial progenitor cells (EPC) are critical in response tovascular injury and repair. EPC levels are reduced in patients with vasculardisease. Pregnancy and diabetes are considered states of endothelial stressand share similar characteristics with vascular disease states. However, thelack of an unique antigen to identify EPCs has lead to different methods ofidentifying EPCs. The widely used Hall method (culture on fibrinogen)identifies non-endothelial hematopoietic cells that nonetheless showcorrelations of decreased numbers with vascular disease. Alternatemethods identify ‘‘late’’ clonogenic EPCs, eg Yoder et al, are of true endo-thelial origin. Using these methods, fetal cord blood levels of EPCs arereduced in diabetes in pregnancy (Ingram et al, Diabetes 57:724, 2008).Objective: To determine if diabetes in pregnancy results in reducedmaternal EPC levels using clonogenic EPC methods.Methods: Human mononuclear cells from whole blood were isolated onFicoll gradients, resuspended in complete EGM-2 medium supplementedwith growth factors and seeded on type I collagen plates. After 24 h, cellswere washed and adherent cells cultured in complete EGM-2-10% FBS withdaily medium changes for 7 d, then every 2 d, until colonies formed.Results: Colony counts at 2 weeks of culture were: normal pregnantwomen: 2.1�0.6 (SEM; n¼23); pregnant diabetic: 1.4�0.3 (n¼24;preeclampsia: 0.8�0.5 (n¼4); normal nonpregnant women: 1.0�0.6(n¼3). ANOVA showed no difference between normal pregnant and dia-betic pregnant women. Preeclampsia and nonpregnant women showedlower colony counts but numbers were insufficient for analysis.Conclusion: Unlike the fetus, true endothelial-derived EPCs are notreduced in the mother with diabetes in pregnancy, suggesting otheretiologies for maternal vascular abnormalities in diabetic pregnancies.Keywords: endothelial progenitor cells, diabetes, preeclampsia

[P06.25].HOMEOBOX GENES ARE DIFFERENTIALLY EXPRESSED IN FETO-PLACENTAL ARTERY AND VENOUS ENDOTHELIAL CELLS OF THEHUMAN PLACENTA

P Murthi*1,2, NA Pathirage1,2, U Hiden3, M Dieber-Rotheneder3, G Desoye3,B Kalionis1,2. 1Department of Obstetrics and Gynaecology, The University ofMelbourne, Australia, 2Department of Perinatal Medicine, PregnancyResearch Centre, The Royal Women’s Hospital, Australia, 3Department ofObstetrics and Gynaecology, Medical University of Graz, Austria

Introduction: Angiogenesis is fundamental to normal placental develop-ment and aberrant angiogenesis contributes substantially to placentalpathologies. The complex process of angiogenesis is regulated by tran-scription factors leading to the formation of endothelial cells that line thevasculature. Homeobox genes are important transcription factors thatregulate vascular development in embryonic and adult tissues. Previousstudies in our laboratory have shown that a number of homeobox genesare expressed in placental endothelial cells (1,2). Recent studies by Lang etal (3) have shown that human feto-placental endothelial cells havea mature arterial and juvenile venous phenotype, which have distinctdifferentiation potentials. In this study, we hypothesised that homeoboxgenes are differentially expressed in the feto-placental artery (HPAEC) andin venous endothelial cells (HPVEC).Methods: HPAEC and HPVEC were obtained from uncomplicated termplacentae as previously described (3). Briefly, HPAEC and HPVEC wereisolated by separate perfusion of chorionic arteries and veins with HBSScontaining 0.1 U/ml collagenase, 0.8 U/ml dispase (Roche), and antibiotics(Gibco). The homeobox gene expression profile of HPAEC and HPVEC wasdetermined using the Human Homeobox (HOX) Genes RT2 Profiler� PCRArray (SAbiosciences) which determined the expression of 84 HOX genesinvolved in multicellular organismal development.Results: Both known and novel homeobox genes showed greater than twofold increased relative expression in HPAEC compared with HPVEC. Knownplacental HOX genes included HLX, HEX, HOXB7, MEIS1, TLX1 and HOXC6.Novel homeobox genes, which showed increased relative expression levelswere CUX1 and HOPX.Discussion: This is the first study to report on differential expression forhomeobox genes in HPAEC and HPVEC. Further analyses on the functionalrole of these homeobox genes in HPAEC and HPVEC maturation anddifferentiation will provide a better understanding of the molecularregulation of placental angiogenesis.References1. Murthi P, et al. Placenta. 2007;28(2-3):219-23.2. Murthi P, et al. Placenta. 2008;29(7):624-30.3. Lang I, et al. Differentiation. 2008;76(10):1031-43.Keywords: Placental endothelial cells, homeobox transcription factors,angiogenesis, gene expression


[P06.26].ANGIOPOIETIN-LIKE PROTEIN 2 (AngPTL2) IN HUMAN MATERNALSERUM, AMNIOTIC FLUID AND TERM PLACENTAL TROPHOBLAST INPREGNANCY

K Naruse*1, M Endo2, A Onogi1, H Shigetomi1, Y Yoshizawa1, T Sado1. 1Dept.of Obstetrics & Gynecology, Nara Medical University, Japan, 2Dept. ofMolecular Genetics, Graduate School of Medical Sciences, KumamotoUniversity, Japan

Introduction: Angiopoietin-like protein 2 (AngPTL2) is a family member ofangiopoietins playing key roles in angiogenesis, vascular maintenance, cellmigration and apoptosis. Although roles of AngPTL2 in tumorgenesis andpathogenesis in tumors and metabolic diseases were defined from formerresearches, little is known about its alteration and expression in pregnancyand placenta. In this study, we measured peripheral AngPTL2 throughpregnancy and searched its expression on cultured trophoblasts separatedfrom human placenta, to assess its possible role in human pregnancy.Methods: Paired serum samples from 1st, 2nd and 3rd trimester of preg-nancy, or prior to and at the next day of selective cesarean section at termfrom respective 7 women were taken at the routine medical examinationwith informed consent. Amniotic fluid samples were taken from 13women at the time of selective cesarean section and centrifuged / filteredbefore the measurement. Serum and amniotic fluid concentrations ofAngPTL2 was measured with ELISA. Human term placentas were taken atselective cesarean section or term vaginal birth. Trophoblast was separatedwith trypsin digestion and Percoll gradient method, harvested on fibro-nectin-coated plate overnight and fixed for staining. Immunocytochem-istry for AngPTL2 and cytokeratin (5+6+8+18) were performed usingrespective antibodies and compared together.Results: AngPTL2 concentration in the serum were significantly increasedin the 3rd trimester of pregnancy (1st, 2.40�0.20; 2nd, 2.93�0.40; 3rd,3.94�0.37 ng/ml, p¼0.0019). Additionally, AngPTL2 in the maternal serumwas significantly decreased at the next day of selective cesarean section(3.45�0.37 vs. 2.41�0.16 ng/ml p¼0.029). Concentration in amniotic fluidwas relatively low (0.06�0.01 ng/ml). Immunostaining of the AngPTL2were positive on the term placental trophoblasts and were matched withcytokeratin-positive cells.Conclusion: Maternal peripheral AngPTL2 increase in pregnant womenwas shown first time, and placenta was suggested as a source of AngPTL2in pregnancy. AngPTL2 may contribute to the placental vascular mainte-nance or other protective roles in the cells at the feto-maternal border.Keywords: AngPTL2, Angiogenesis, Placental vascular maintenance, feto-maternal border

[P06.27].METHOTREXATE AND EPIDERMAL GROWTH FACTOR INHIBITIONREGRESS PLACENTAL-DERIVED TISSUES IN VITRO AND IN VIVO:TOWARDS A NOVEL COMBINATION MEDICAL THERAPY TO TREATECTOPIC PREGNANCY

UW Nilsson*1,2, YE Gao1,2, TG Johns1, BR Williams1, ED Williams1, S Tong1,2.1Centre for Cancer Research, Monash Institute of Medical Research,Australia, 2Centre for Women’s Health Research, Monash Institute ofMedical Research, Australia

Introduction: Ectopic pregnancies are serious gynaecological emergenciesthat can cause fatal haemorrhage. Most (75%) require surgery. Systemicmethotrexate (MTX) is sometimes used. Since efficacy is patchy, it onlyworks for small ectopics.We hypothesise that Epidermal Growth Factor Receptor (EGFR) inhibition� MTX could efficaciously resolve stable ectopic pregnancies of any size.The placenta relies on the EGFR pathway for survival. EGFR inhibitors,currently on the market to treat cancers, have few side effects. Further-more, EGFR inhibitors have supra-additive anti-tumour activity whencombined with chemotherapeutics.We set out to develop a novel approach to medically treat ectopic preg-nancy using the EGFR inhibitor gefitinib (Iressa�) � MTX.Methods/Results: In vitro: We first confirmed by immunostaining that1st trimester trophoblast, BeWo and JEG-3 cells express EGFR. Next, weadded various treatments to placental-derived cells and assayed viability72 hours later (CellTiter-Blue [Promega]). Gefitinib alone did not causetrophoblast cell death of JEG-3 (see figure) or BeWo cells (not shown)compared to control. MTX alone (1-1000uM) decreased cell viability ina dose-responsive manner (not shown). Interestingly, when gefitinibwas combined with MTX, a dose-dependent, supra-additive decrease incell viability occurred in both JEG-3 (figure A) and BeWo cells (notshown).In vivo: SCID mice (n¼3-5 per group) were inoculated with JEG-3 cells(106), and treated with different doses of gefitinib and MTX, all as singleagents (see figure). Compared to vehicle-treated mice, there was a signifi-cant inhibition of xenograft growth with both doses of gefitinib where onlyw30% had palpable tumours by 19 days. There was also a dose dependentinhibition of xenograft growth with MTX.Conclusion: Gefitinib � MTX causes significant regression of placentalderived tissues. It may be a novel therapeutic approach to treat stableectopic pregnancies medically where conceivably, tablets (Gefitinib +MTX) could replace surgery.

Keywords: Ectopic pregnancy, Epidermal growth factor, Methotrexate,Therapy


[P06.28].RNA INTERFERENCE-INCUCED REDUCTION IN ASCT2 EXPRESSION,A SYNCYTIN RECEPTOR, SUPPRESSES CELL FUSION OF HUMANPLACENTAL BeWo CELLS

T Nobuzane*, S Matsuyama, Y Kudo. Department of Obstetrics andGynecology, Hiroshima University, Japan

Introduction: Syncytin, the human endogenous retrovirus envelopeproteins, is highly expressed in the placental syncytiotrophoblast layer,and contributes to placental fusion events. ASCT2 is a syncytin receptorand known to be identified as the amino acid transporter B0. The decreaseof fusion activity in hypoxia was observed. Additionally the alteration ofthese gene expressions in pre-eclampsia has been reported. To investigatethe relationships between syncytin, ASCT2 and fusion activity, we usesiRNA technique in human placental BeWo cells.Methods: BeWo cells were treated with siRNA for either syncytin orASCT2. The efficacy of these specific siRNA treatments was analyzed andcell fusion was assessed by flow-cytometry.Results: The mRNA expression of syncytin and ASCT2 was reduced aftersiRNA treatment. The fusion activity was reduced only after treatmentwith siRNA for ASCT2.Discussion: These results suggest that ASCT2 might be involved in theprocess of cell fusion in BeWo cells.Keywords: cell fusion, ASCT2, BeWo cells

[P06.30].DETERMINATION OF THE FIRST LINEAGES DURING CATTLEEMBRYOGENESIS

D. Berg, C. Smith*, D. Wells, R. Broadhurst, M. Berg, P.L. Pfeffer. Agresearch,New Zealand

The first lineage decision during mammalian embryogenesis is betweeninner cell mass (ICM) and trophectoderm (TE). By blastocyst stages theselineages are clearly discernable morphologically. In mice, ICM and TErequire the function, and are characterised by the mutually exclusiveexpression, of Oct4 (ICM) and Cdx2 (TE). This has led to models implicatingthese factors in early lineage determination. We were thus surprised tofind only marginal lineage-specific enrichment of these genes in cattleblastocysts, using quantitative PCR. Lineage tracing confirmed that blas-tocyst TE cells were fated to remain trophectodermal. However, sand-wiching Day7 blastocyst or Day14 gastrulation-stage TE cells withinmorula cells revealed that blastocyst TE was not yet committed. Wespeculated that lack of TE commitment in cattle was linked to the observedretarded downregulation of Oct4 in these cells. We could demonstrate thatmouse and cattle Oct4 regulation is fundamentally different at blastocyststages. Unlike mouse embryos, cattle embryos transgenically modified (vianuclear transfer) with mouse Oct4 regulatory sequences driving a GFPreporter were unable to shut off the mouse Oct4 reporter at blastocyststages. Conversely, both transgenic mouse and cattle blastocysts wereunable to restrict a cattle Oct4 reporter to the ICM. These experimentsindicate that not only does the cattle embryo lack the necessary factorspresent in the mouse for shutting off Oct4 transcription in the blastocyst TEcompartment, but that the cattle Oct4 gene is missing the ‘mouse-TE-specific’ negative regulatory elements. Was Cdx2 responsible for Oct4downregulation? pCAG-GFP-AntiCdx2-miR mediated knock-down of Cdx2expression had no effect on Oct4 transcription but led to severe TE defectsat gastrulation stages. The rodent-ruminant differences in timing of bothcommitment and Oct4/Cdx2 lineage restriction may be adaptations todifferent requirements: whereas mouse embryos implant as blastocysts,cattle embryos are less hasty, attaching only post-gastrulation.Keywords: lineage determination, bovine, trophectoderm, preimplantation


[P06.31].INSULIN INCREASE D-GLUCOSE TRANSPORT IN HUMAN UMBILICALVEIN ENDOTHELIAL CELLS

C Puebla*, CA Sepulveda, JL Vega, P Casanello, L Sobrevia. Cellular andMolecular Physiology Laboratory (CMPL) and Perinatology ResearchLaboratory (PRL), Department of Obstetrics & Gynaecology, School ofMedicine, Pontificia Universidad Catolica de Chile, Chile

Introduction: Umbilical cordon represent the medium to communicationbetween mother and fetus. Nutrients are in contact directly with theendothelium, which is affected by the different concentrations of thesemolecules. In endothelial cells, facilitative and insulin-insensitive trans-porters are the principal protagonist in the transport of D-glucose, but it iscontroversially discussed wheter the endothelium represents an insulin-responsive tissue, inclusive the insulin-sensitive GLUT4 isoform seems tobe expressed in human umbilical vein endothelial cells (HUVEC) fromnormal pregnancies. We studied if D-glucose transport in HUVEC fromnormal pregnancies exposed to high D-glucose is modulated by insulin.Methods: HUVEC were isolated by collagenase (0,2 mg/ml) from humanumbilical cord vein from normal pregnancies and exposed (24 hours) to 5mM (normal) or 25 mM (high) D-glucose in absence or presence of insulin(10 nM, 8 hours). 2-deoxi-D-[3H]glucose (2DG) transport (1,6 mM, 1 mCi/ml, 5 sec to 40 sec, 22�C) was measured.Results: 2DG transport is lower in HUVEC exposed (24 h) to high D-glucose. Then in presence of insulin the 2DG transport increase in HUVECexposed to high D-glucose, but in normal D-glucose the effect was absent.Discussion: D-glucose transport is decreased in HUVEC exposed to high D-glucose, but in presence of insulin this transport is increased to normalcondition, which could be by the presence of insulin-sensible transporterGLUT-4; eventually this phenomenon is a compensatory effect: cells try todecreased extracellular glucose levels to protect the fetus. The fact thatinsulin did increase D-glucose transport only in cells exposed to high D-glucose, could suggest that this pathological environmental conditioncould increase insulin-sensitivity for GLUT-4.FONDECYT 1070865/1080534/7070249 (Chile), C Puebla and JL Vega holdCONICYT PhD fellowships.Keywords: Endothelium, Transport, Glucose

[P06.32].ENDOCRINE GLAND-DERIVED VASCULAR ENDOTHELIAL GROWTHFACTOR (EG-VEGF) EXPRESSION IN TWO TRANSIENT ORGANS:PLACENTA AND THYMUS OF DOGS – A COMPARATIVE STUDY

Fernanda R. Agreste*1, Patrıcia R. Moriconi1, Bruno Cogliati1, Pedro P.Bombonato1, Carlos Eduardo Ambrosio1, Christiane Pfarrer2. 1Faculty ofVeterinary Medicine and Animal Sciences, University of Sao Paulo, Brazil,2University of Veterinary Medicine Hannover, Germany

Recently, EG-VEGF (endocrine gland-derived vascular endothelial growthfactor) was described in human and mouse placenta and is likely to playan important role in placentation as it stimulates proliferation, migra-tion, and fenestration selectively in capillary endothelial cells fromendocrine glands. However, the EG-VEGF mRNA level determined inothers organs like liver and kidney, suggests that EG-VEGF may havedirect effects in these tissues and thus is not restricted to endocrinetissues. Thymus and placenta are transient organs showing commonfeatures of elevated metabolic activity and modifications in cellularproliferation and differentiation when undergoing continuous stress. Incontrast to most domestic species the dog placenta does not producesteroid hormones.Our aim was therefore to localize and quantify EG-VEGF throughoutdevelopment and initial involution of two transient organs, canine thymusand placenta. Thymuses from 30, 40, 50 and 60 days-old fetuses, 6 and 12months-old dogs (n¼6 per group) and dog placentae from 20, 40 and 60days of gestation (n¼4 per group) were examined by immunohisto-chemistry and Western blot.EG-VEGF was detected in all stages of development and involution inthymus and placenta. In thymus, protein was localized in medullar endo-thelial and epithelial cells while no staining occurred in the cortical region.In placenta, EG-VEGF was detected mainly in endothelial cells and syn-cytiotrophoblast of labyrinth.Protein quantification revealed higher (p<0.05) values during develop-ment of thymus (days 30-60) and placenta (days 20-40), decreasing at theend of gestation for the placenta. During thymus involution (6-12 months)EG-VEGF levels were constant but lower (p<0.05) than during gestation.Our results suggest that EG-VEGF may play important roles during thymusand placenta development and involution, which have to be specified infurther studies. Possible functions include modulatory effects on thymicand placental microenvironment, influencing cellular proliferation,differentiation and secretion. Funded by CAPES, FAPESP and DAAD.Keywords: placenta, EG-VEGF, canine, thymus


[P06.33].UNALTERED 5-HYDROXYTYPTAMINE (5-HT) RECEPTOR EXPRESSION INPLACENTA, DESPITE INCREASED CIRCULATING 5HT IN PRE-ECLAMPSIA

S. D. Sivasubramaniam*, P. Muralitharan, L. De Girolamo. Nottingham TrentUniversity, United Kingdom

This study aimed to investigate the status of circulating 5HT and the mRNAexpressions of three main vascular 5-hydroxytryptamine (5-HT or sero-tonin) 1B, 2A and 7 receptors in normotensive (NT) and pre-eclamptic (PE)placentae.5HT mainly produces vasoconstriction in the peripheral vasculature and inplacental vessels. Previous studies have demonstrated that 5-HT can playroles in placental development and pregnancy maintenance. It may alsotake part in regulating fetal development. Since the effects of 5-HT arebrought about by 7 different types (and their subtypes) of 5-HT receptors,it would be interesting to study the status/expression of these receptors inplacental tissues.Circulating levels of 5-HT were indirectly determined by the analysis ofurinary 5-HT and its metabolite 5-hydroxyindoleacetate (5-HIAA) byELISA. The status and the expression of 5-HT receptors between NT and PEplacentae were compared by RT-PCR and quantitative real time (qRT-PCR)respectively in relation to two house-keeping genes GAPDH and b-actin.Pre-eclamptic women had significantly higher levels of 5-HT(124.8�23.6mg/24h urine) compared to NT counterparts (59.5�9.8mg/24hurine) (p<0.05).Whilst the levels of the metabolite 5-HIAA were similar inthe two groups. Thus the ratio of 5-HIAA:5-HT in PE women was 50% lowerthan NT controls (p<0.05). RT-PCR results have shown the presence of5HT1B, 2A and 7 receptors in NT and pre-eclamptic samples. However wehave found there are no significant differences in the mRNA expressions ofthese receptors between NT and PE placentae. For example the 5HT2Aexpression in relation to b-actin in NT and PE placentae were 4.40(SEM�1.24) and 4.02 (SEM�1.27) respectively. Similar patterns wereobserved for 5HT1B and 5HT7 receptor expressions.These results suggest that despite a definite increase in the circulating 5-HT levels in PE, there is no change in the mRNA expressions of 5HT1B, 2Aand 7 receptors in PE placentae.Keywords: 5-hydroxytryptamine, pre-eclampsia, qRT-PCR

[P06.34].CLINICAL ANALYSES FOR TRANSITIONAL CASES OF INFERTILITY ANDRECURRENT PREGNANCY LOSS

E.T. Takahashi*, R.K. Kawaguchi, T.T. Tadao. Department of Obstetrics andGynecology, The Jikei University, School of Medicine., Japan

Objectives: In the clinical practice, it is not a little case that the patient whoexperience recurrent pregnancy loss (RPL) becomes to be infertilitythereafter, or tends to be aborted repeatedly after infertile therapy. Thosetransitional conditions have not been noticed so far.We investigated the difference of possible causes and clinical status amongthose conditions from perspectives of reproductive failure.Patients and Methods: Out of patients of infertility (694) and RPL (691)treated at The Jikei University Hospital from 2003 – 2008, 4 groups wereselected as follows.A : transitional cases from RPL to infertility (24).B : transitional cases from infertility to RPL (43).C : cases of infertility (40).D : cases of RPL (80).Results: 1. Although the ratio of pregnancy did not differ significantlyamong 4 Groups, that of miscarriage showed higher in A and B than C andD, and moreover that of live-born showed the fewest in A compared withD, also with significant difference.2. The incidence of positive for APAs approximately amounted to about 50percent of these factors in all Groups. Especially, in B, APAs was recognizedsignificantly higher in elderly-age than young-age (under 35 years).3. The reproductive outcome after therapies was examined in all Groups.Concerning infertility, C showed significantly fewer rate of miscarriagethan A and B regardless of the therapies, ART or not. On the other hand,although anti-coagulant therapy was treated for RPL, D showed signifi-cantly fewer rate of miscarriage than A and B.Conclusion: 1. The state of reproductive failure in transitional cases havea tendency to be unfavorable reproductive outcome.2. In transitional cases of reproductive failure, active treatment such as ARTis recommended from the early period.Keywords: anti-phospholipid antibody, infertility, recurrent pregnancyloss


[P06.35].THERAPEUTIC OUTCOME IN RECURRENT SPONTANEOUS ABORTIONSWITH ANTIPHOSPHOLIPID ANTIBODIES(aPLs). w THE INFLUENCE OFTITERS, VARIETIES, ISOTYPES, POSITIVE NUMBERS OFANTIPHOSPHOLIPID ANTIBODIES

N Umehara*, R Kawaguchi, S Wada, K Sugiura, K Ooura, T Tanaka.Department of Obstetrics and Gynecology, The Jikei University School ofMedicine, Japan

Objective: Low dose heparin in combination with low dose aspirin is thestandard therapy for patients with histories of recurrent pregnancy losses andpositive for antiphospholipid antibodies, but the influence of antibody class,titer, isotype of IgG and IgM, positive number of the aPLs are not yet clear.Our objective is to investigate the reproductive outcome of anticoagulanttherapy to patients of recurrent spontenous abortion with anti-phospholipid antibody, with special attention to the impact of species,titer, and isotype of the antibody.Method: I had a checkup 282 examples with histories of two or morespontenous abortions, visited in this hospital from April, 2000 to July,2005, and detected serum levels in 10 varieties of aPLs. Of 156 patients ofrecurrent abortion with antiphospholipid antibody, 101 cases got pregnantand anticoagulant therapy was performed according to our anti-coagulation protocol, so these 101 cases were evaluated. Furthermore, weclassified them into low and high titer groups, IgG or IgM isotypes groups,single positive or plural positive groups with aPLs. In each groups, weanalyzed successful rate of pregnancy prospectively.Results: Species, titer or isotypes of antiphospholipid antibodies did notaffect the rate of maintained pregnancy in recurrent abortion when anti-coagulant therapy was actively introduced. Regarding on isotypes and thenumber of positive antibodies, aspirin treatment alone was sufficient forsuccessful reproductive outcome in only the IgM-isotype and single anti-body positive cases (95.2%), while in the IgG-isotype and plural antibodiespositive cases, the rate of maintained pregnancy was increased bycombination treatment with aspirin and heparin (95.2%) than aspirinalone (78.6%).Conclusions: The anticoagulant therapy was effective to patients ofrecurrent abortion with antiphospholipid. However, combination therapywith aspirin and heparin may be overtreatment for only the IgM-isotypepositive case.Keywords: recurrent spontaneous abortion, antiphopholipid antibodies,reproductive outcomes, anticoagulant therapy

[P06.36].MONOCHORIONIC TWIN FETUSES SHOWING A REVERSAL OF DONOR-RECIPIENT PHENOTYPES IN SEVERE TWIN–TWIN TRANSFUSIONSYNDROME WITHOUT OLIGO-POLYHYDRAMNIOS SEQUENCE

A Mitsumori*, O Akutagawa, M Kazuka, H Funayama, K Isaka. TokyoMedical University, Japan

Antenatal sonographic diagnosis of twin–twin transfusion syndrome(TTTS) is based on findings of a twin oligo-polyhydramnios sequence(TOPS) observed in monochorionic twin fetuses. However, TTTS candevelop without a significant characteristic intertwin discordance in theamniotic fluid volumes. We present an uncommon form of TTTS withoutTOPS showing severe anemia in one twin and polycythemia in the other.A 34-year-old Japanese woman was referred to our hospital at 30 weeks ofgestation for perinatal management of a monochorionic-diamniotic twinpregnancy. Although no abnormalities were noted by ultrasound exami-nation before her first visit to our hospital.Both fetuses had a normally filled bladder and a normal amniotic fluidvolume. The patient was admitted for close perinatal management at 30weeks. Because of increased uterine activity, the intravenous administra-tion of ritodrine hydrochloride was initiated.At 34 weeks, the fetal heart rate monitoring twin 1 showed loss of vari-ability. Because of non-reassuring fetal status of twin 1, weighed 2341 gand was pale, whereas infant 2 weighed 1920 g and was plethoric. Apgarscore at 1 and 5 minute after birth were 2/4 and 6/6 for infant 1 and 2,respectively. And hemoglobin contents were 4.7g/dl and 20.3g/dl.The placenta was monochorionic diamniotic, with a velamentous insertionof the edematous umbilical cord of twin 2. The placental territory of twin 1was pale, three times larger than that of twin 2, which was plethoric.In conclusion, perinatologists involved in the care of monochorionic twinsshould keep the differential diagnosis of acute or chronic TTTS in mind,even in the absence of TOPS. TAPS can occur in the absence of the char-acteristic interwin discordance of the amniotic fluid volume.

[P07.01].PLACENTAL AND FOETAL ORGAN GROWTH ARE REGULATED BYDIFFERENT COMBINATIONS OF GROWTH FACTORS

R. S. F. Lee*, N. Li, D. N. Wells. AgResearch, New Zealand

The cloning of cattle by somatic cell nuclear transfer (SCNT) is characterizedby increased incidence of abnormal development and excessive placentaland foetal growth. In SCNT foetuses from a Friesian dairy cow cell line, wefound no apparent correlation between placental and foetal weight, sug-gesting that placental and foetal growth may be independently regulated.We examined the expression of several growth-regulating genes, such asIGF2, H19, IGF2R, IGFBP-2, GPC3 and CDKN1C (p57kip2), in mid-gestationSCNT placenta and foetal tissues that normally show overgrowth (liver,kidney and heart) using northern blots. SCNT samples were compared withsamples from gestation-matched half-siblings generated by artificialinsemination (AI) or from the transfer of in vitro produced (IVP) embryos.The mean expression levels for IGF2, H19, IGF2R and GPC3 in either foetal orplacental samples were not different among the SCNT, AI or IVP groups.Mean IGFBP-2 expression was significantly lower (P<0.05) in the SCNT andIVP placenta compared with the AI controls, consistent with IGFBP-2 beinga negative regulator of IGF-II action and the observed placental overgrowthin SCNT, and to a lesser extent, IVP. No difference was detected in kidney andheart samples but mean expression in SCNT liver tended to be higher. Incontrast, there was no difference in the expression of CDKN1C in placental,liver and heart tissues. Mean expression in the kidney was significantlylower (P<0.001) in SCNT, where there was a significant inverse correlation(P<0.005) between the level of CDKN1C expression and kidney weight.Thus, the growth of each foetal organ and the placenta is regulated bydifferent combinations of factors that act in an autocrine/paracrinemanner rather than systemically. In SCNT foetuses, this may not be as wellcoordinated between the different organs and placenta resulting in loss ofallometric growth regulation.Keywords: IGFBP-2, CDKN1C, SCNT, overgrowth


[P07.02].ENDOCRINE GLAND-DERIVED VASCULAR ENDOTHELIAL GROWTHFACTOR (EG-VEGF) EXPRESSION IN CLONED AND NON-CLONEDBOVINE PLACENTA

FR Agreste1, LA Fatima1, C Pfarrer2, PC Papa*1. 1Sector of Anatomy,Department of Surgery, Faculty of Veterinary Medicine and AnimalSciences, University of Sao Paulo, Sao Paulo, Brazil, 2Department ofAnatomy, University of Veterinary Medicine Hannover, Germany

Endocrine gland vascular endothelial growth factor (EG-VEGF), also knownas prokineticin 1 (PK1), has recently been described in the human andmouse placenta. It promotes proliferation, survival, and chemotaxis ofendothelial cells in steroidogenic tissues, such as adrenal cortex capillaryendothelial cells. The bovine placenta is multiplex, villous and synepi-theliochorial due to migratory trophoblast giant cells (TGC).To determine the spatio-temporal distribution of EG-VEGF in non-clonedand cloned bovine placenta, placentomes from non-cloned pregnanciesfrom early gestation until near term (6 groups: days 30, 60, 90, 150, 210 and270 of gestation, n¼4 per group) and cloned placenta near term (n¼4)were evaluated by immunohistochemistry and quantified by western blot.EG-VEGF immunoreactivity was observed in uterine glands duringimplantation. In normal and cloned bovine placenta the immunoreactionwas confined to maternal epithelium, binucleate TGC, fetal and maternalblood vessels. The quantification of EG-VEGF in normal bovine placentarevealed that high levels occurred in early placentation; these decreasedfrom mid gestation (p<0.05) until near term. In cloned bovine placentaethe expression was higher (p<0.05) than in non-cloned placentae.In summary, EG-VEGF is present in the synepitheliochorial bovine placentain all studied gestational phases but the levels are related to gestationalage. Thus, we may conclude that EG-VEGF expression in the bovineplacenta seems to be important throughout gestation, probably influ-encing endothelial and/or epithelial cells function. Moreover, placentaderived from cloned gestations, which presented documented differencesregarding vascularization pattern and expression of vascular relatedgrowth factors, showed an up-regulation of EG-VEGF expression, regard-less of fetus gender. This up-regulation could reflect the attempt tocompensate hypoxia in these placentae.Funded by CAPES, FAPESP and DAAD.Keywords: EG-VEGF, bovine, SCNT

[P07.03].EXPRESSION OF ALPHA6BETA1 AND ALPHAVBETA3 INTEGRINS ANDTHEIR EXTRACELLULAR MATRIX LIGANDS IN PLACENTOMES FROMCLONED AND NON-CLONED CALVES

LP Artoni1, N Hambruch2, PC Papa1, C Pfarrer*2. 1Institute of Anatomy,Department of Surgery, University of Sao Paulo, Sao Paulo, Brazil,2Department of Anatomy, University of Veterinary Medicine Hannover,Hannover, Germany

Integrins are transmembrane glycoproteins which are involved in cell-celland cell-extracellular matrix (ECM) adhesion and signal transduction.Integrins may be involved in the migration and fusion of trophoblast giantcells (TGC) with uterine epithelial cells in bovine placentomes. Somatic cellnuclear transfer is frequently associated with aberrant placentation aslarger placenta with lower number of placentomes, hydroallantois andlarge offspring syndrome. Since integrins are considered to be constitutiveproteins being responsible for the maintenance of the architecture withintissues, we aimed to verify a potential relation of integrins with commonplacental alterations observed in cloned animals.Bovine placentomes were collected and divided into 3 groups: 1) mid and2) late gestation derived from natural mating (n¼9) and 3) late gestationalbovine clones (n¼7). ECM proteins Collagen IV, Fibronectin and Lamininwere localized by indirect immunohistochemistry and integrin subunitsalpha6, beta1, alphaV and beta3 were shown by immunofluorescence. Theantibody specificity was confirmed by Western blot.In cloned and non-cloned animals the integrin subunits alpha6 and beta1were observed near the basal membrane of maternal and fetal epithelialcells and stromal endothelium, and co-localized with laminin in some TGC.The integrin subunits alphaV and beta3 were co-localized with thecollagen IV and fibronectin in maternal and fetal stroma. Western blotconfirmed the integrin and laminin antibodies specificity. No difference inthe integrin or extracellular matrix proteins expression could be foundbetween mid and late gestational placentomes from natural matingpregnancies and cloned gestation.The absence of differences found between cloned and non-cloned gesta-tion suggests that the interaction of integrins with their ECM ligandscannot be directly related with the observed placental alterations.Nevertheless, further studies are necessary to elucidate the mechanismsthat might be responsible for the alterations observed in the gestation ofbovine clones.Funded by CAPES, FAPESP and DAAD.Keywords: Integrins, bovine, SCNT


[P08.01].ORGANOGENESIS OF THE CARDIO-RESPIRATORY SYSTEM OF BOVINEEMBRYOS MANIPULATED IN LABORATORY

M. L. V. Alberto*, F. V. Meirelles, A. C. R. Galdos, R. Ricci, A. G. T. Pessolato,M. A. Miglino. Medical College of Zootechny and Veterinary Medicine,Section of Domestic and Wild Animals Anatomy USP/SP, Brazil

Morphogenic changes of cardio-respiratory system of cattle from in vitrofertilization and nuclear transfer are the main factors responsible for highincidence of embryo, fetal and postnatal mortality. The study of develop-ment of heart and lung was made using techniques of light microscopy. Inthe embryos derived from in vitro fertilization at 28 days of gestation wasfound the laryngotracheal tube and its septation through the fold trache-oesophageal. In this same period, the embryos showed pericardial cavity,atrium divided into right and left portions, cone heart, venous sinus,myocardium and epicardium layer. Was observed sprouting in the rightlateral portion of the trachea, cranial to its bifurcation in embryos with 36days of gestational age. At 44 days of gestation the lung buds of theembryos showed the main bronchi originated of segmental bronchi. Themesenchyma of support in differentiation contained blood vesselsdispersed, unlike embryos from nuclear transfer. These embryos at 68 daysof gestation showed lung in pseudoglandular phase containing bronchiolesbuds and absence of blood vessels. At 70 days of gestation, the heart of thefetus showed significantly large ventricle, small lobby and undevelopedlung. The placenta is considered a vital organ of pregnancy. Thereforea flaw in the formation and development process can cause seriouschanges in organogenesis of the embryo and growth of the fetus until birth.Keywords: cardio-respiratory system, bovine embryos, lung buds, heart

[P08.02].LOOKING FOR THE VITELLINE PLACENTATION IN EARLY BOVINESEMBRYOS

Celina Almeida Furlanetto Mançanares, Ana Flavia de Carvalho, RudolfLeiser, Carlos Eduardo Ambrosio, Maria Angelica Miglino*. 1UNIfeob, SaoJoao da Boa Vista, Sao Paulo, Brazil, 2Department of Veterinary Anatomy,Histology and Embryology, Justus –Liebig-University Giessen, Germany,3University of Sao Paulo, FMVZ, Surgery Department, Brazil

Yolk sac is an important organ to embryonic circulation and metabolicprocess mainly during the transition of chorionvitteline placentationprocess to chorionallantois one. About 25% to 40% of embryo lost occursduring fertilization process until implantation. The yolk sac is the mainlysource of nutrition before true placenta will completely formed and yourimportance are related to hematopoiesis. The aim of this work is describesthe primitive gut transitional area with yolk sac connection and maintainimportance to the embryo until placental formation. Were collected 59embryos during 10 to 50 days of pregnancy and were grouped by Crown-rump measure and external characteristics, fixed, and processed byprocedures for embedding in paraffin. Sections were stained by HE. Theembryo yolk sac was observed with active cells between it connectionwith primitive gut at anterior portion. Gut cells presented at begin ofdifferentiation of epithelium columnar cells and endoderm follow downby undifferentiated layer of mesenchymal cells from the mesoderm. Grossaspect of this fetal membrane is centrally compact with two free elon-gated tips at 10 to 20 days of pregnancy and became decreases it size upto 40 days after fecundation. The yolk sac was formed by blood vesselsisland surrounding to endodermic cells and mesenchyme. Also was foundnucleated blood cells (hemangioblast) from fetal origin. Mesenchymallayer was thin with elongated cell characterized by proteoglycansynthesis presented into cell matrix. At transitional strait channelbetween yolk sac and primitive gut with delicate roundest cells and intothe lumen was found hemangioblast follow in fetus way direction andalso coming into the mesenchyme. Although this last tissue type presenthemangioblast into small capillary net. So, how is the real function of yolksac and the vitelline nutrition marked the first environment adversity ofthe embryo life?Keywords: development embryonary, yolk sac, vitelline placentation

[P08.03].INITIAL DEVELOPMENT OF BOVINE PLACENTATION (BOS INDICUS)FROM THE POINT OF VIEW OF THE ALLANTOIS AND AMNION

A. C. Assis Neto*1, C. E. Ambrosio2, M. F. Oliveira3, F. T. V. Pereira1, M. A.Miglino2. 1Sao Paulo State University, Brazil, 2University of Sao Paulo,Brazil, 3Universidade Federal Rural do Semiarido, Brazil, 4Sao Paulo StateUniversity, Brazil, 5University of Sao Paulo, Brazil

The aim of this study was to perform a morphological characterization ofthe initial bovine placental development, from 20 and 70 days post-insemination (p.i), with emphasis on the differentiation of the allantois andamnion. After collection, the conceptuses were dissected, macroscopicallymeasured and photographically documented. The extraembryonicmembranes were cut into fragments measuring 5 cm2, and afterwards fixedin 4% paraformoldehyde for analysis by light microscopy, and 2.5% glutar-aldehyde for use in scanning and transmission electron microscopy. Theextraembryonic and fetal membranes presented variable degrees ofdevelopment throughout the periods analyzed. The macroscopic appear-ance of vascularization of the allantois, its attempt to merge with the cho-rium, and the effective appearance of the first cotyledons in developmentwere events observed from 30 to 40 days of pregnancy. The measurementsof the amnion increased gradually as gestation developed. The allantoicepithelia presented cellular dimorphism from 20 to 25 days of pregnancy,but was shown to be immature from 60 to 70 days of pregnancy.Financial support: FAPESP, CAPES, CNPq, FUNDUNESPKeywords: Placenta, Allantois, Amnio, Bovine

[P08.04].PLACENTAL GROWTH IN MINK: ELUCIDATED BY MITOSIS, APOPTOSISAND TURNOVER RATE THROUGH OUT GESTATION

Vibeke Dantzer*, Henrik Winther. 1IBHV, LIFE, Copenhagen University,Denmark, 2BImmunoHistology, Dako A/S, Produktionsvej 42, DK-2600Glostrup, Denmark

Objective: The development of placenta, growth and angiogenesis isa prerequest for successful gestation. We here demonstrate the spatio- andtemporal localization of the mitotic activity, caspase activity and endo-thelial turnover rate in a small carnivore, the mink, having an incompletezonary, villous, endothelio-chorial placenta and delayed implantation.Methods: Gestational age was estimated by uterine dilations (ampullae)and the fetal crown/rump (C/R) length (term 7cm). By immunohisto-chemical the activity of Ki-67 and Caspase-3 activity (18 mink) and 11mink, injected with bromodeoxyuridin (Br-dUrd) 24-168 hrs beforeeutansia, was studied.Results: Ki-67 activityat nonpregnant was only seen in the stroma. At invasionthe endometrium was very active also in glandular epithelium being lower inthe luminal epithelium. Trophoblast cells were very active and especiallyduring primary invasion, where the apposing maternal tissue except, vascularendothelium become non-reactive, this invasion pattern was seen up to 50-60mm here close to the glandular part. The activity in glandular epitheliumremained almost to late stage. The maternal endothelium was active to aboutlate mid gestation and then declined rapidly showing only few mitotic cells at60 mm, however the mitotic activity was dominated by trophoblast and fetalvasculature declining later than the activity in the maternal endotheliumCaspase activity was high in tissues apposed to invasion.Conclusion: 1) early mink placentation is characterized by high mitoticactivity in the invasive trophoblast and uterine glands; 2) growth is high tolate mid gestation mainly at the fetal placental side 3) maternal endothelialcell turnover rate at mid gestation is 5-7 days.Keywords: Mink, mustelidae, placental growth pattern, Mitosis, Apoptosis


[P08.05].MATERNAL DIETARY ARGININE SUPPLEMENTATION DURINGGESTATION IMPROVES REPRODUCTIVE EFFICIENCY IN PIGS

MJ De Blasio*1, JA Owens1, CT Roberts1, R Smits2, M Nottle1. 1The RobinsonInstitute & School of Paediatrics and Reproductive Health, University ofAdelaide, Australia, 2QAF Meat Industries Pty Ltd, Corowa NSW, Australia

Introduction: Arginine (a non-essential amino acid) and its conversion tonitric oxide (NO) by nitric oxide synthase (NOS) can regulate the formation ofnew blood vessels and vasodilation. This may reduce resistance and increaseblood flow to the uterus and placenta, and hence delivery of nutrients for fetalgrowth and survival. In rats, arginine deficiency causes IUGR and increasesfetal death and perinatal mortality, whereas dietary arginine supplementa-tion reverses this. Human IUGR is associated with impaired NO synthesis,arginine transport, and eNOS activity in umbilical vein endothelial cells, butsupplementing with arginine has produced inconclusive results.We hypothesised that maternal arginine supplementation (MAS) in the pig(a species with naturally occurring IUGR due to a large litter size) duringearly gestation, when placental angiogenesis and vascularity increase,would increase birth weight and placental weight.Methods: Gilts (parity 0) and sows (parity 3) (n¼150/parity) were feda control or arginine supplemented (+25g/d arginine) diet (2.5kg/d), inearly gestation (17-33d). Birth and placental weights of progeny weremeasured at term (115d).Results: MAS reduced pregnancy loss in both parities and increasednumber of progeny born alive (Control vs. Arginine: 10.7 vs. 11.9, +1.2p¼0.048) in primiparous pigs. MAS increased litter birth weight (12.3 vs.14 kg, +1.7kg p<0.05) and litter placental weight (2.63 vs. 3.93kg, +1.3kgp<0.05) in parity 0, but not parity 3 pigs.Discussion: Maternal arginine supplementation during critical periods ofplacental development may enhance placental-fetal blood flow andnutrient transfer, thereby improving fetal growth and survival andameliorate the effects of IUGR.Keywords: Arginine supplementation, early gestation, pigs

[P08.06].EQUINE PLACENTA BY SCANNING ELECTRON MICROSCOPY (SEM)

Ana Flavia de Carvalho, Priscila Leal do Nascimento, Andre Luis RezendeFranciolli*. 1Dept. of Morphology, UNIfeob, Sao Joao da Boa Vista, Brazil,2Dept. of Surgery, Faculty of Veterinary Medicine, University of Sao Paulo,Brazil, 3Presbiteriana Mackenzie University, Brazil

The epitheliochorial placentation in horses is result of placenta develop-ment. The aim of this research was to analyze the morphology using SEMof the equine embryonic membranes, to provide key parameters related tothe equine reproduction. We used 13 placentas (0-120 days of gestation)fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer pH 7.4. The chorionwith less than 36 days of gestation showed chorionic projections with thecellular apical surface rounded and heptagon shape and covered bymicrovilli, with 3.1 cm of CR (36 days of gestation) round cellular surfaceswithout microvilli (smooth chorion). The chorion with 6.7 cm to 12.3 cm ofCR (57 to 79 days of gestation), showed numerous round projections, withgoffer shape to endometrial direction. The chorion with 10.3 cm to 20.2 cmof CR (71 to 107 days of gestation) showed ultrastructural features similarto animals with 6.7 cm to 12.3 cm of CR. This showed a hexagonal cell, apexwith a few trophoblast cells without microvilli and others with microvilliof different sizes. The allantoic membrane in all gestational periodsobserved showed characteristic slightly rough, with microvilli in the cellssurface forming images pentagon, hexagonal and heptagon. Round struc-tures were observed with "button" shape, suggesting adhesion betweenboth chorion and allantoic. The amnion had a similar to the allantoic,whose cells varied in tetragonal to heptagonal shape it is delimited bymicrovilli strings. In the amniotic surface were observed a number ofstructures with severe shapes, similar to ‘‘buttons’’, as observed in allantoicmembrane. The yolk sac in all groups showed droplets secretion in the cellapex showing exocytose, and its surface was irregular due to the secretiongranules. In conclusion, we believe that equine embryonic membrane issimilar to other artiodactyls membranes as pigs, ruminants and camelids.Keywords: placenta, equine, SEM, fetal membranes

[P08.07].STUDY OF THE EQUINE PLACENTA UP TO 120 DAYS OF GESTATION INLIGHT MICROSCOPY (LM) AND TRANSMITION ELECTRON MICROSCOPY(TEM)

Ana Flavia de Carvalho1, Ana Claudia Cristiane Ferraz1, Priscila Leal doNascimento1, Celina Almeida Furlanetto Mançanares1, Ana CarolinaFurlanetto Mançanares1, Andre Luis Rezende Franciolli*2. 1Dept. ofMorphology, UNIfeob, Sao Joao da Boa Vista, Sao Paulo, Brazil, 2Dept. ofSurgery, Faculty of Veterinary Medicine, University of Sao Paulo, Sao Paulo,Brazil

The epitheliochorial placentation in horses is a result of placenta devel-opment. The aim of this study was to morphologic analyze equineembryonic membranes under LM and TEM, to provide key parametersrelated to the equine reproduction. It was observed 43 placentas (0-120days of gestation) fixed in 10.0% formaldehyde plus 2.5% glutaraldehyde in0.1M phosphate buffer (pH 7.4), and processed by paraffin embedding andHE, tricromic Masson and PAS reaction. Under LM, trophoblast in earlygestation (36 days) presented few villous with columnar low epitheliumlayer and brush border, uni or binuclear cells, that multiply rapidly andacquire an invasive phenotype, basal membrane and a highly vascular-izated conjunctive tissue. As gestation proceeded, the villous became morecomplex revealing primary villous on days 53-88. The alantoic membranepresented squamous and mesenchymal cells, full of vessels, fusioned tochorion forming the chorioalantoic membrane. The amnions presentedsquamous cells, supported by mesenchymal cells, fusioned with amnionresulted in amniochorion membrane. Yolk sac had large round cells, sup-ported by mesenchymal cells with hemangioblasts islands and mesothelialboundries. In TEM, chorion presented epithelium columnar cells (uni, bi ormultinuclear cells), mesenquimal tissue rich in collagen fibers, formingtrophoblast. A large amount of mitochondria in cytoplasm of cellular apexand eletrondense vesicles was observed. Allantoic cells presented roundnuclei and subdivided cytoplasm, a high concentration of mitochondriasand endoplasmic granular reticulum in the cellular apex or base, respec-tively. The amnion presented an only layer of flattened cells supported byconjunctive tissue. Also, it was observed a large amount of eletrondensegranules in the cellular apex surrounded by a round shape shadow. Inconclusion, we suggested that equine embryonic and fetal membrane issimilar to other artiodactyls membranes as in pigs, ruminants (cattle andsheep) and camelids (llamas).Keywords: placenta, Early palcentation in mare, morphologic aspects


[P08.08].CATHEPSIN B, CATHEPSIN L AND CYSTATIN C IN PORCINE UTERI ANDPLACENTAE: A MODEL FOR PROTEIN MODIFICATION DURINGUTILIZATION AND FLUID-PHASE TRANSPORT ACROSS UTERINEEPITHELIA, AREOLAE AND NEONATAL GUT

G.A. Johnson*1, G. Song2, D.W. Bailey1, K.A. Dunlap1, R.C. Burghardt1, F.W.Bazer2. 1Department of Veterinary Integrative Biosciences, Texas A&MUniversity, United States, 2Department of Animal Science, Texas A&MUniversity, United States

Proteases, Cathepsins (CTSB) and (CTSL1), and their inhibitors, Cystatin C(CST3), remodel endometrium and placenta for transplacental transport ofgases, micronutrients and macromolecules. We examined CTSB, CTSL1 andCST3 expression and hormonal regulation in endometria and placentae ofpigs.1) gilts were hysterectomized on Day 9,12, or 15 of the estrous cycle, orDay 9,12,15, 20, 25, 30, 35, 40, 50, 60, or 85 of pregnancy. 2) cyclic gilts wereinjected (Days 11-14) with estrogen (E2) and hysterectomized on Day 15 or90 of pseudopregnancy. 3) gilts were ovariectomized on Day 12, injectedwith progesterone (P4) for 28 days, and hysterectomized on Day 40. CTSBincreased in luminal epithelium (LE) after Day 30 of gestation. CTSB in LEwas not affected by E2, but was increased by P4. CTSB was abundant inchorionic epithelium by Day 20 and remained through Day 85. CTSL1increased in LE, GE and trophectoderm by Day 20 of gestation. EndometrialCTSL1 was not affected by E2, but was increased by P4. CTSL1 was highlyexpressed in the chorionic epithelia that form areolae to absorb secretionsof uterine glands. CST3 was expressed in presumptive immune cells withinendometria, but increased in LE by Day 25 of gestation. CST3 was notaffected by E2 at Day 15, but increased in LE on Day 90 of pseudopregnancy.P4 decreased CST3 in LE, but increased expression in immune cells. We nextexamined CTSL1 in the jejunum of neonatal pigs which absorbs IgG forpassive immunity. CTSL1 was expressed in enterocytes of villi. These resultsshow that CTSL1 is expressed by areolae, GE, and neonatal intestine, each ofwhich have roles in fluid-phase transport of proteins, and that interactionsbetween CTSL1, CST3 and CTSB may modify proteins during utilization andtransport across uterine, placental and gut epithelia.Keywords: Pig, proteases, areolae, uterus

[P08.09].CAVEOLAE AND CAVEOLINS IN THE CLONED TRANSGENIC CATTLEPLACENTA

FTV Pereira*1, FV Meirelles2, F Bressan2, M Miranda2, AC Assis Neto1, MAMiglino2. 1Paulista State University, Brazil, 2University of Sao Paulo, Brazil

Caveolae and caveolin-mediated endocytosis are internalization pathways inthe placental transport. However, their roles in a cattle placenta have notbeen addressed. In this study, we compared the involvement of caveolae- andcaveolins -1,-2 in cloned transgenic cattle. Fetal fibroblasts expressing theGFP gene were used as nuclei donors to cloning by nuclear transfer (NT), toproduce the gestations by embryo transfer. Transmission electron micros-copy (TEM) and immunhistochemistry (IHC – anti-caveolins -1, -2) wereperformed on placentomes and chorioallantois from 5 cloned (60 and 90days of gestation) and 10 controls in the same gestation period. The tissueswere glutaraldehyde or formalin fixed. At the TEM we could observe andcharacterize the structures called caveolae in blood capillaries of thechorioallantoic membrane and placentomes by natural (control) and clonedtransgenic cattle gestation. The caveolae appears as a plasmaleme vesiclesand invaginations in both of the plasmic membrane in luminal and abluminalportion. However, we have observed many microvilli in the trophoblast ofthe placentome, such as in the chorioallantoic membrane. The fetal andmaternal villi were immunoreactive to caveolin-1, but the strongest reactionwas observed in the endometrial stroma. The chorioallantois trophoblast andplacentome were immunoreactive to caveolin -2, mainly in a trophoblastgiant binucleate cell. The results obtained by the TEM and IHC indicated thatthe caveolae plus microvilli are very important sites on placental transportand signaling in the natural and cloned transgenic cattle gestation.Funded: FAPESP.Keywords: Caveolae, Caveolins, Cloned cattle, Transgenic

[P08.10].EXPRESSION AND MODULATION OF Wnt, Bmp, AND PUTATIVEANTAGONIST GENES IN THE BOVINE CARUNCLE ENDOMETRIUMDURING THE IMPLANTATION STAGE

M B Aires1, K Y Degaki1, V Dantzer2, T E Spencer3, A T Yamada*1. 1Universityof Campinas, Brazil, 2Faculty of Life Sciences, Denmark, 3Texas A&MUniversity, United States

Development of villous-like projection on cow caruncle (CAR) surface aslocal endometrium response to establish the embryo-chorion anchoragesite for synepithelialchorial placentation, closely resembles epithelial-mesenchyme interaction during embryo organogenesis of gut, urinay andrespiratory mucosa that are strictly controlled by morphogens like WNTand BMP proteins families. The present work comparatively evaluated theevolution of villous-like projection in the pregnant and non-pregnat (NP)cow (Bos spp) caruncle based on morphological analysis and, Wnt and Bmpmorphogen gene families expression by PCR and in situ hybridization. Byhistological analysis of CAR mucosa during implantation was establishedfour developmental stages: S1–trophoblast cells uterine epitheliumadhesion, S2– initial villous-like projections on the CAR surface, S3–expansion and anastomoses of villous-like projections, and S4– pla-centome consolidation. Anti-PCNA immunostaining demonstrated intenseproliferative activity from S1toS4 on CAR epithelial and stromal cells,resulting in a 5-fold expansion of endometrial tissue. By RT-PCR and in situhybridization, the expression of the Bmp2 and Wnt7a genes was seen up-regulated in the CAR, with the highest expression level detected at S1compared to NP. Otherwise, Wnt2 and Wnt5a expression levels were lowerin the CAR. Dkk1 and Sfrp2 were widely expressed in the LP and S1-S4 CARand IC 45 endometrium. Noggin expression was consistently lower in theCAR, whereas Sostdc1 expression was higher in the CAR than IC at S1 andthen declined in both regions until S4. Therefore, simultaneous expressionof Wnt and Bmp together with their antagonists in the CAR and IC regionsseems to be responsible for the capability of cow endometrium quickremodeling and ability to develop synepithelialchorial placentation.Furthermore, they also attest to the complex synergistic/antagonisticsignaling mechanisms of several still unknown factors modulating the cowuterine mucosa response during embryo implantation and synepithe-lialchorial placentation. Grants: CNPq and CAPES.Keywords: placentome, caruncle endometrium, morphogen, WNT andBMP


[P09.01].PLACENTAL IGFBP-1, IGF-I, IGF-II AND THE MICRONUTRIENTS ZINC ANDFERRITIN IN THE PAKISTANI SGA POPULATION

Shahzad Akram*1, Christine Carlsson-Skwirut1, Zulfiqar Bhutta2, OlleSoder1. 1Karolinska Institutet, Sweden, 2The Aga khan University, Pakistan

Background: The insulin-like growth factor (IGF) axis plays an importantrole in normal gestational development. Alterations in this axis can resultin serious consequences, such as small for gestational age (SGA) babies.Micronutrient supplementation, namely zinc, has been shown to decreasethe risks of preterm pregnancy and may thus regulate this axis. OBJECTIVE:The principal objective of this study was to correlate placental levels of theIGF-axis, namely, IGFBP-1 with IGF-I and IGF-II, and the micronutrientszinc and ferritin, in SGA and large for gestational age (LGA) babies in thePakistani population.Methods: Human placental samples were obtained from 89 women inrural field sites in Pakistan. Samples were divided into SGA and LGA groupsbased on population percentiles.Results: Higher levels of IGFBP-1 were seen in the SGA group as comparedto the LGA group (p<0.01). Multivariate regression for IGFBP-1 levelssignificantly correlated with mRNA expression levels of IGF-II in the SGAgroup (p<0.05). No significant correlations were seen with IGF-I expres-sion, zinc levels, or ferritin levels.Conclusion: We have shown the important differences in IGFBP-1 levels inplacenta, comparing SGA and LGA groups. This suggests the key role thisprotein plays in growth regulation, particularly towards the end of gesta-tion. Though no significance was seen when analysing the binding proteinwith respect to the micronutrients, the correlations suggest their role inregulation of IGFBP-1. The importance of a well-balanced system cannot bestressed enough, as both deficiencies and excessive amounts of IGFBP-1can tip the balance of growth in either direction.Keywords: SGA, IGFBP-1, IGF-I, micronutrients

[P09.02].IGF-I, OESTROGEN RECEPTOR, AND PROGESTERONE RECEPTOREXPRESSION, AND MATERNAL ANTHROPOMETRY IN GROWTHRESTRICTED PREGNANCIES: A LOOK AT THE SWEDISH POPULATION

Shahzad Akram*, Lena Sahlin, Ewa Ostlund, Gabriel Fried (Late), Olle Soder.Karolinska Institutet, Sweden

Background: Foetal growth restriction is a complex problem of pregnancy,arising from multiple aetiologies, including environmental, nutritional andgenetic problems. A key regulatory element of cellular and tissue growth,at the endocrine level, is the insulin-like growth factor (IGF) axis. Bothexcesses and deficiencies in this axis contribute to foetal growth problems,the most common outcome being babies being born small for theirgestational age (SGA).Aim: To determine the relations of placental levels of IGF-I, oestrogenreceptor (OR), and progesterone receptor (PR) expression with maternalanthropometry, looking at birth weight outcomes.Methods: Placental samples were obtained from 33 patients, followingdelivery, from the Karolinska Hospital, Stockholm, Sweden. Experimentalmethods included hybridisation probe analysis for nucleic acids and PCRtechniques.Preliminary Results: Placental expression of IGF-I, OR, and PR were alldecreased in the SGA group, compared to the NC group. However, onlythose of IGF-I were statistically significant (p<0.05). Furthermore, nosignificant correlation was seen between maternal anthropometry and theabove mentioned factors.Conclusion: The differences in placental expression of emphasises the keyrole the growth factor and receptors play in birth weight outcomes,particularly in babies born SGA. The expression of these receptors maythus potentially be used as markers for SGA. Though maternal anthro-pometry has been seen to contribute to birth weight outcomes, throughmaternal constraint, it did not appear to be a significant contributing factorin the Swedish population.Keywords: IGF-I, IUGR, PR, OR


[P09.04].PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR g (PPARg)MEDIATED MECHANISM FOR PLACENTAL APOPTOSIS IN MATERNALFOOD RESTRICTED GESTATIONS

L Belkacemi*, MG Ross, Q Liu, M Desai. Department of Obstetrics andGynecology, David Geffen School of Medicine at UCLA and LABIOMEDResearch Institute at Harbor-UCLA Medical Center, CA, USA, United States

Introduction: Maternal food restriction (MFR) during pregnancy leads tointrauterine growth restricted (IUGR) fetuses that have a higher risk ofadult obesity, hypertension and diabetes. IUGR may occur secondary toinsufficient maternal nutrient supply and/or reduced placental nutrient/oxygen transfer. Notably, increased placental apoptosis has been associ-ated with IUGR. The mechanism may involve peroxisome proliferator-activated receptor g (PPARg) which is an anti-apoptotic transcriptionfactor and mediates cellular proliferation and differentiation. We haveshown that MFR placentas have reduced growth at gestational ages, E16and E20. Therefore, we investigated the impact of MFR on placentalapoptosis and PPARg expression at ages, E16 and E20. We focused ourstudy on the two placental positions (proximal and mid-horn) with theextremes of nutrient/oxygen supply, and two distinct placental zones(basal, site of hormone production; and labyrinth, site of feto-maternalexchange).Methods: Pregnant rat dams were fed an ad libitum diet (AdLib) or were50% MFR beginning at E10 of gestation. At E16 and E20, fetal and placentaswere recorded. Six placentas from left proximal and mid-horns were usedfor TUNEL staining. The corresponding right mid- and proximal hornplacentas were separated into basal and labyrinth zones and analyzed forzone and region specific expression of PPARg (Western blot).Results: MFR-induced fetal growth restriction was evident only at E20. Incontrast at E16, as compared to AdLib, the MFR mid-horn placentas hadreduced basal zone and similar labyrinth zone weights. No weight changeswere seen in proximal horn placentas. By At E20, MFR mid- and proximalhorn placentas showed significant reduction in the basal and labyrinthzones. Interestingly, the level of apoptosis was significantly increased inthe MFR from mid- and proximal horn placentas at E16 and E20. Consistentwith this, PPARg protein expression was significantly downregulated atboth E16 and E20 in the MFR placentas.Conclusion: The placentas show early impact (E16) of MFR as evident byincreased apoptosis and reduced growth. This in turn may contribute tofetal growth restriction in MFR pregnancies. Further, MFR inducedplacental apoptosis may, in part, be mediated by PPARg.Keywords: apoptosis, IUGR, placenta, PPARg

[P09.05].DIFFERENTIAL REGULATION OF GROWTH AND GENE EXPRESSION OFCULTURED HTR-8/SVneo HUMAN TROPHOBLASTS BY NUTRIENTRESTRICTION AND HYPOXIA

P.M. Brannon*, S. Jones, J.Y. Lee. Cornell University, United States

Maternal undernutrition (characterized by restricted energy, protein andmicronutrient intake) decreases placental and fetal growth throughunknown mechanisms. Hypoxia (Hx) and reduced nutrient availabilitysubsequent to reduced placental blood flow are proposed mediators. Weinvestigated the effects of NR and Hx on growth, gene expression and hCGsecretion in the HTR-8/SVneo human trophoblast cell line (Tb). Cells (1- 6 x105) were plated in 75 cm2 flasks in RPMI media with 1.25%-5% FBS andcultured with 1-20% oxygen. We determined cell number by trypan blueexclusion or spectrofluorometric DNA analysis, protein by Lowry, mRNA byRT-PCR and hCG by ELISA. Data from 3 replicate experiments with 3-4samples/treatment were analyzed by ANOVA or 2-way ANOVA. Growthwas log-linear with 1-4 x 105 plated cells for 96 h; 2 x 105 cells were platedfor subsequent experiments. A 75% reduction (NR) of media glucose,essential amino acids and Gln, and vitamins reduced growth 52% at 72 h.Reduced oxygen (1, 2 or 4%) decreased growth at 72 h by 10-53% comparedto 8 or 20% oxygen. Glut 3 mRNA increased even with 8% oxygen (1 fold)and maximally increased 7 fold with 1% oxygen; HIF1a and Ki67 mRNAdecreased dose responsively to a minimum in cells grown at 1% oxygen(0.6 and 0.5 fold, respectively); VEGF mRNA increased biphasically witha maximum (1.4 fold) at 2% oxygen. Because mRNA responses occurredwith 8% oxygen, 20% oxygen was selected as the ‘normoxic’ level; 1%oxygen was selected as Hx because it resulted in maximal change inseveral mRNA’s. In a 2x2 factorial experiment at 72 h, Hx, independent ofNR, decreased growth 43.5%; NR independent of Hx decreased growth34%; and Hx X NR interacted such that Hx only increased VEGF mRNA incomplete media (1 fold). Hx, but not NR, independently increased glut3mRNA (1 fold), Igf2 mRNA (0.62 fold), cell size (protein/DNA ratio, 141%)and secreted hCG (242%). Hx and NR both independently, but not inter-actively decreased the growth of Tb. The differential effect of Hx on glut3and Igf2 mRNA and secreted hCG also suggests Hx acts independently toregulate some gene expression. However, the absence of an effect of Hx onVEGF mRNA in NR suggests that Hx and NR may also interact through oneor more regulatory pathways.Keywords: maternal malnutriton, nutrient restriction, hypoxia, growth


[P09.07].TRANSPLACENTAL GLUCOSE TRANSPORT IN PLACENTAL MALARIA

C.L.L. Chua*1, A.J. Umbers1, E. Chaluluka2, S.J. Rogerson1, P. Boeuf1.1University of Melbourne, Australia, 2University of Malawi, Malawi

Each year, 50 million pregnancies are at risk of malaria. Placental malariainfection is associated with fetal growth restriction, resulting in low birthweight babies being born to infected mothers. The pathogenetic mecha-nisms in placental malaria which lead to compromised fetal growth areunknown. However, the sequestration of malaria parasites in the placentaoften results in an inflammatory response being initiated in the inter-villous spaces (intervillositis). These events cause placental structuraldamages and may impair placental functions, including nutrient transport.Given that transplacental glucose transport is an essential factor control-ling fetal growth, we hypothesised that impairment in glucose transportmay be a potential mechanism underlying fetal growth restriction inplacental malaria.Using placental samples from Malawi, which consisted of malaria-infectedplacentas with or without intervillositis, we quantified the expression ofthe glucose transporter GLUT-1 in the syncytiotrophoblast microvillousand basal membranes. GLUT-1 expression profiles were then compared tothat obtained on uninfected placentas from the same region. In this pilotstudy, GLUT-1 expression in the basal membrane was lower in the malaria-infected placentas with intervillositis than in those without intervillositis(P¼0.064) or control placentas (P¼0.021).Given that GLUT-1 expression in the basal membrane is the limiting factorfor transplacental glucose transport, we propose that the inflammatoryresponse associated with placental malaria has an adverse effect on theplacental ability to transport glucose to the fetus. Our results suggest thatmalaria-induced placental intervillositis can cause fetal growth restrictionthrough decreased nutrient transfer and shed new lights on the patho-genesis of fetal growth restriction during malaria in pregnancy.Keywords: Placental malaria, fetal growth restriction, intervillositis,GLUT-1 expression

[P09.08].ROLE OF MICRORNAS IN PLACENTAL PROGRAMMING OF INSULINRESISTANCE

H. Harryanto*, M.L. Harland, P.A. Grant, M.J. De Blasio, J.S. Robinson, J.A.Owens. The University of Adelaide, Research Centre for the Early OriginsHealth and Diseases, The Robinson Intitute, Australia

Introduction: Placental restriction (PR) is a major cause of intrauterinegrowth restriction (IUGR) which is associated with adult-onset type 2diabetes. This is due to insulin resistance and down-regulation of geneand/or protein expression of insulin signalling molecules in skeletalmuscle in human, rats and sheep. MicroRNAs (miRs) are small non proteincoding RNAs that can down-regulate of multiple targets mRNAs. Wehypothesised that PR and IUGR in the sheep alters expression of miRs inskeletal muscle of progeny, which able to alter insulin signalling expres-sion and its action, in lambs (44 days old) and adults (18 months old).Methods: Placental growth in sheep was restricted by removal of themajority of endomentrial caruncles from non-pregnant Merino ewes, andhence placental size and function. Vastus lateralis was collected at post-mortem, miR expression analysed by Exiqon microarray v8.1. Bio-informatic analyses used to identify the predicted gene targets for eachdifferentially expressed miR. Ingenuity Pathway Analysis (IPA) was used toidentify metabolic and signalling and other pathways that might beaffected.Results: PR reduced skeletal muscle expression of miR-211 and miR-451(p<0.05) in lambs overall and only miR-365 (p<0.05) in females and didnot alter any in males. In adult sheep, PR increased miR expression infemales only, they are miR-278, miR-376b, miR-175p, miR-142-3p, miR-21,miR-101 and miR-324-3p (p<0.05). Finally, PR reduced skeletal muscleexpression of miRs in male progeny, regardless of age; including miR-663,miR-711, miR-720, miR-612, miR-197, miR-409-5p and miR-769-3p(p<0.05). Some of them were predicted to target mRNAs of proximalinsulin signalling molecules, and other pathways; ERK/MAPK, GABAreceptor and JAK/Stat signalling.Conclusion: The present study shows that early life perturbation by PR canalter the expression of miRs in skeletal muscle of progeny and lead to thealtered expression of insulin signalling molecules and potentially othersignalling pathways.Keywords: IUGR, microRNA, insulin resistance, skeletal muscle


[P09.09].HIF-1 TRANSCRIPTIONAL ACTIVITY IS INDUCED RAPIDLY BY HYPOXIAIN PRIMARY SYNCYTIOTROPHOBLAST

N.P. Illsley*, E. Fik, S. Zamudio. UMDNJ-New Jersey Medical School,United States

The HIF-1 transcription factor is a central mediator of the cellular responseto hypoxia. We hypothesized that there would be a time-dependentincrease in HIF-1 transcriptional activity in human syncytial cells inresponse to hypoxia. To define the HIF-1 transcriptional profile we trans-fected trophoblast cells with a HIF-luciferase reporter and measured theresponse to a hypoxia-mimetic, dimethyloxallylglycine (DMOG), aninhibitor of HIF-1a degradation.Methods: Cytotrophoblast were allowed to plate for 18 hr before incuba-tion with lentiviral particles (low or high titer) expressing the luciferasegene under the control of hypoxia response elements. After 48 hr incu-bation, cells were switched to medium in the absence (control) or presence(treated) of 1 mM DMOG. Triplicate samples were assayed for luciferaseactivity following 2, 6 or 18 hr of incubation.Results: Luciferase activity in the control cells was similar over the threetime points however it was 3.0 � 0.3 fold higher in cells subjected totreatment with the lower viral titer; we report here the response in thelow titer-treated cells. At 2 hr, luciferase activity was 3.7 � 0.6 fold higherin treated compared to control cells. After incubations of 6 hr and 18 hr,activity in treated cells was 2.6 � 0.3 and 2.1 � 0.2 fold higher.Conclusions: These results show the feasibility of lentiviral transductionalthough the reduced level of luciferase activity in cells transfected witha higher viral titer suggests that cytotoxicity may be an importantconsideration. The response to DMOG in these initial experiments wassurprising in that there was a rapid response at the early time point fol-lowed by a decrease to level 2-fold higher than control by 18 hr. This datasuggests that these cells can respond to hypoxia in both an acute andchronic manner. Supported by HD46982 (NI) and HD42737 (SZ).Keywords: HIF-1, Hypoxia, Syncytiotrophoblast, Human

[P09.10].PLACENTAL ANTIOXIDANT EXPRESSION IN SHEEP FOLLOWINGPLACENTAL INSUFFICIENCY

N.E. Marshall*, P. O’Tierney, S. Louey, K.L. Thornburg. Oregon Health andScience University, United States

Introduction: Antioxidants play a key role in scavenging reactive oxygenspecies (ROS) generated during normal metabolic processes. Disruption ofthe balance between free radical production and antioxidant activity (e.g.by intermittent hypoxia and reoxygenation) leads to oxidative stress andcellular damage. Placental insufficiency from daily umbilicoplacentalembolization (UPE) in sheep leads to fetal hypoxemia which is intermittentduring the first 5-7d of embolization. We hypothesized that the cellularstress associated with embolization would stimulate the expression ofantioxidant system protein.Methods: Fetal sheep underwent daily UPE from 115 days gestation (termw145d) to a level that leads to growth restriction. After 5 days of UPE,placental samples from UPE (n¼6) and control fetuses (n¼6) were snapfrozen in liquid nitrogen. Superoxide dismutase (SOD), glutathioneperoxidase (GPx), and catalase mRNA expression were measured in UPEand control cotyledons using real time quantitative PCR; expression wasstandardized to GAPDH as ratios, data are mean�SEM.Results: UPE fetuses were mildly hypoxemic during the study (PaO2:15.3�0.7 vs 20.7�0.4mmHg, p<0.01). No statistically significant differ-ences in antioxidant protein message expression were found. SODs cata-lyze the conversion of superoxide to hydrogen peroxide. SOD1 expressionwas 1.1�0.6 (UPE) vs 2.7�1.7 (control), p>0.40; SOD2 expression was1.2�0.3 (UPE) vs 0.7�0.1 (control), p>0.10. GPx1, GPx4 and catalaseconvert hydrogen peroxide to oxygen and water. GPx4 expression was1.9�0.6 (UPE) vs 1.1�0.2 (control), p>0.20 and even lower averageexpression of GPx1 1.1�0.3 (UPE) vs 2.0�0.6 (control), p>0.20; catalaseexpression was 2.0�0.9 (UPE) vs 4.4�2.8 (control), p>0.40.Conclusion: Despite significant and intermittent fetal hypoxemia,placental antioxidant expression levels were not significantly altered byplacental insufficiency. Differing levels of hypoxemia and recovery fromembolization may account for the considerable variation in antioxidantexpression. We are now studying the effects of a longer period of placentalinsufficiency on placental antioxidant expression and activity.Keywords: oxidative stress, antioxidant, placental insufficiency


[P09.11].THE ROLE OF NODAL IN THE UTERUS DURING PREGNANCY

C. B. Park*, D. Dufort. McGill University, Canada

Pre-term birth (PTB) is the leading cause of perinatal mortality, accountingfor over 75% of perinatal death. Despite recent progress, PTB has continuedto rise over the years and remains an important clinical dilemma in bothdeveloping and developed countries. Failure to decrease the rates of PTB isattributed, in part, to our lack of understanding of the causes and mech-anisms that underlie pre-term delivery. In order to aid in the ongoingpursuit of elucidating these mechanisms, our laboratory has been char-acterizing the expression of the TGF-b superfamily member, Nodal, in theuterus and investigating the potential role this factor may play in facili-tating the birth of healthy offspring.Utilizing the loxP-Cre recombinase system, we have generated a condi-tional knockout of Nodal in the female reproductive tract of adult mice,bypassing embryonic lethality. Interestingly, the Nodal deficient miceexhibit various reproductive abnormalities, including reduced rates ofestablishing pregnancy, intrauterine growth restriction (IUGR) and fetalabortion late into development (d17.5). The placenta of the Nodal condi-tional knockout mothers exhibit significant expansion of the labyrinth, andmarked reduction of the spongiotrophoblast and maternal decidual layers.Furthermore, the trophoblast giant cells appear disorganized and signifi-cantly reduced in number.We report here, the first phenotypic characterization of a uterine Nodalknockout strain, implicating the Nodal signalling pathway in facilitatinghealthy pregnancy. Our observations indicate that Nodal ligand froma maternal source may play a crucial role in decidualization and properplacenta development and its absence leads to IUGR, PTB and aborteddevelopment. Understanding the mechanisms that underlie IUGR and pre-term delivery are paramount in the ultimate goal of eliminating compli-cations during pregnancy leading to pre-eclampsia, PTB and embryonicloss.Keywords: Nodal, Pre-Term Birth, Intrauterine Growth Restriction

[P09.12].REDUCED VASCULARITY OF THE FETOPLACENTAL ARTERIAL TREE INTRANSGENIC MICE WITH FETAL HEPATIC OVEREXPRESSION OFhIGFBP-1

MY Rennie*1,4, KJ Whiteley2, CS Watson2, VKM Han3, SL Adamson2,5,JG Sled1,4. 1Mouse Imaging Centre, Hospital for Sick Children, Canada,2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Canada,3Children’s Health Research Institute, University of Western Ontario,Canada, 4Dept. of Medical Biophysics, University of Toronto, Canada,5Depts of Physiology and Ob/Gyn, University of Toronto, Canada

Introduction: Insulin-like growth factors (IGFs) are critical to fetal andplacental growth and their actions are inhibited by IGF binding proteins(IGFBPs). IGFBP-1 is elevated in the circulation of human IUGR newborns.As in human IUGR, transgenic mice (Tg) with elevated circulating hIGFBP-1are growth-restricted and have abnormal umbilical artery Doppler wave-forms, suggesting alterations in the fetoplacental vascular tree. Thepurpose of this study was to quantify the arterial fetoplacental vasculaturein this model.Methods: Fetoplacental arterial trees from wild type (WT) and Tg micewere infused with X-ray contrast agent at embryonic day 15.5 and 3-Dmicro-CT images were obtained. Custom designed image processingsoftware was used to determine length, diameter, and connectivity of eacharterial segment more than 50 mm in diameter.Results: Fetal weight was reduced 21% in Tg fetuses. Tg vascular trees weremore often asymmetric (5/10) compared to controls (0/12), with increasedvascular depth but decreased span. Umbilical artery diameter wasunchanged, however Tg trees had w20% fewer vessel segments in bothsmall (50-100 mm) and large (>100 mm) vessel diameter ranges.

WT (n¼12)
Tg (n¼10)
Fetal weight (g)
0.41 � 0.01 0.33 � 0.01* Placental weight (g) 0.15 � 0.01 0.14 � 0.01 Vascular span (mm) 6.79 � 0.11 6.45 � 0.10* Vascular depth (mm) 1.13 � 0.02 1.30 � 0.05* Umbilical artery diameter (mm) 0.49 � 0.06 0.46 � 0.03 Segments 50-100mm 1310 � 46 1101 � 51* Segments >100mm 178 � 9 152 � 6*
*p<0.05 (t-test). Values shown as mean � SEM.

Conclusions: Elevated hIGFBP-1 in Tg mice causes fetal growth restrictionand an abnormally shaped arterial fetoplacental tree with significantlyfewer vessels. Therefore IUGR due to elevated circulating IGFBP-1 may bedue in part to reduced fetoplacental vascularity and impaired placentalfunction. Supported by Heart and Stroke Foundation of Ontario.Keywords: fetoplacental vasculature, IGFBP-1, micro-computed tomog-raphy, IUGR


[P09.13].PLACENTAL PLASTICITY IN A NON-HUMAN PRIMATE MODEL OFPLACENTAL INSUFFICIENCY: GESTATIONAL TIMING IS CRITICAL TOADAPTIVE CAPABILITY

VHJ Roberts*, JP Rasanen, MJ Novy, KE Sparks, S Louey, KL Thornburg.Oregon Health and Science University, United States

Introduction: Structural abnormalities of the placenta or impaired bloodsupply lead to poor fetal outcomes including IUGR. The hemochorialplacenta of the rhesus monkey is analogous to the human placenta withthe added feature of a secondary lobe. Ligation of the bridging vessels toeliminate the functional capacity of the secondary lobe has been used asa model of placental insufficiency (AJOG 2009; 199 suppl: 6A #430). Ouraim was to determine the effects of ligation in early vs late gestation onplacental growth regulating factors and volume blood flow (QPL).Methods: Inter-placental bridging vessels were surgically ligated at either80 days gestation (Early, n¼3) or 110 days (Late, n¼3 term¼167 days).Doppler ultrasound measurements were conducted to correlate changes inQPL and fetal growth indices before and after ligation. Tissues were har-vested from experimental animals and age-matched controls at 140 days,and placental characteristics recorded. Placental protein expression ofmTOR, VEGF and 11bHSD2 was determined by western blot.Results: Functional placental mass (Control; combined lobes 85.0�4.0gSEM) increased with early ligation (primary lobe 96.97�5.5g) anddecreased with late ligation (primary lobe 75.47�7.9g). Similarly QPL

increased in the early group and was diminished in the late group. Fetalweights were reduced in both groups compared to control (Early: 340�2g;Late: 329�3g; Control: 419�3g). A 30% increase in placental mTORexpression was demonstrated in the early group compared to control,whilst VEGF and 11bHSD2 expression remained unchanged in both groups.Conclusion: The diminished capacity of the placenta to compensate fora vascular insult in late compared to early gestation suggests a criticaldevelopmental window for plasticity. The mTOR pathway may play a keyrole in signaling the compensatory mechanisms which maintain thenutrient supply of the fetus.Keywords: Rhesus monkey, Placental insufficency, Placental plasticity, mTOR

[P09.14].DOES PLACENTAL WEIGHT RELATIVE TO BIRTHWEIGHT AFFECTOUTCOME FOR STILLBIRTHS AND NEONATAL DEATHS?

JMD Thompson*. University of Auckland, New Zealand

Introduction: The placenta is an integral part of pregnancy and the growthof the fetus. The aim of this study was to determine whether so called‘‘placental insufficiency’’ is related to the outcomes of birthweight andplacental weight, and whether the effect on these outcomes is propor-tionate in the presence of conditions resulting in stillbirth or neonataldeath.Methods: Data on birthweight and placental weight was obtained for alldeliveries at National Womens Hospital, Auckland from 1993-2000(n¼69,680), and the ratio of the two calculated. These were converted to z-scores according to population norms and were analysed according tocause of death (PSANZ definitions) for stillbirths (n¼437) and neonataldeaths (n¼410) in comparison to all live births.Results: Compared to normal controls, stillbirths due to congenitalanomalies were significant lighter whilst their placental weights werenormal. For stillbirths associated with hypertension, and IUGR placentalweight and birthweight were affected proportionately, whilst deathscaused by spontaneous preterm birth and unexplained deaths had rela-tively normal birthweights and placental weights.For neonatal deaths placental weights were generally proportionatelyhigher than birthweights for most groups of death but particularly thosedeaths due to extreme prematurity, cardio-respiratory disorders, andgastro-intestinal disorders.Conclusions: Placental function as defined by size is often cited as a causeof poor outcomes despite the fact that one would expect a normal distri-bution of placental weights for any given birthweight. This study showsthat whilst for some causes of death birthweight is affected proportion-ately to placental weight, for others birthweight is below that which wouldbe expected from the placental weight. This suggests that ‘‘placentalinsufficiency’’ may not be the issue in relation to poor outcomes but thatother mechanisms such as utero-placental blood flow or compensatorygrowth may be important in some causes of death.Keywords: Placental weight, placental function, fetal death


[P09.15].MATERNAL SERUM LEVELS OF ANGIOPOIETIN-2 (ANG-2) IN IUGRPREGNANCIES, AND ANG-2 LOCALISATION IN REPRODUCTIVE TISSUES

Y Wang*1, C Woolnough1,2, V Tasevski1,2, E Gallery1, J Morris1. 1PerinatalResearch group, Kolling Institute of Medical Research, University ofSydney, Australia, 2Fetal Maternal Medicine (PaLMS), Royal North ShoreHospital, Australia

Introduction: Reduced serum levels of Ang-2 were observed in pregnantwomen at 10-13 weeks gestation whose pregnancies were subsequentlycomplicated by intrauterine growth restriction (IUGR)1. The aims of thisstudy were: (1) to describe serum levels of Ang-2 throughout menstrualcycle and during normal pregnancy, (2) to compare serum Ang-2 levelsbetween IUGR and normal controls at the time IUGR was first diagnosed,and (3) to immunolocalise Ang-2 in placenta, placental bed, and deciduafrom both normal and/or IUGR pregnanciesMethods: 29 non-pregnant and 68 normal pregnant women at distincttime points were recruited. Serum samples were also collected from 17IUGR patients and their paired normal controls. Ang-2 was measured usingan R&D systems ELISA assay. Chorionic villi were obtained from diagnosticchorionic villous sampling (CVS) at 10-13 weeks. Placenta and deciduawere collected at the time of delivery from both normal and IUGR subjects.Protein localisation of Ang-2 was examined using immunohistochemistry.Results: Serum levels of Ang-2 were similar between the follicular andluteal phases. Levels of Ang-2 rose at 8 weeks, peaked at 13 weeks, anddeclined from 18 weeks of gestation. The median concentration of Ang-2 atthe 13-week peak was 18.71 ng/mL, compared to 1.72 ng/mL in non-pregnant women (p<0.01) (Figure 1). Serum levels of Ang-2 were signif-icantly lower in IUGR pregnancies than normal controls at diagnosis.(p<0.05). Ang-2 tissue expression was found in the syncytiotrophoblast ofboth the first trimester (CVS) and the third trimester placentas. Ang-2expression was also found in invasive cytotrophoblast, endothelial anddecidual cells. (Figure 2).

Figure 1. Serum levels of Ang-2 throughout transverse the menstrual cycle and duringpregnancy.

Figure 2. Ang-2 localisation in a section of placenta.

Discussion: These findings suggest that Ang-2 potentially plays importantroles in early placental angiogenesis, with a 10-fold increase at 8 weeks ofgestation. Placental angiogenesis may be compromised in IUGR pregnancy.Cells of fetal and maternal origin contribute to high Ang-2 serum levels inpregnancy.Reference1. Wang et al., BJOG 2007.Keywords: Angiopoietin-2, IUGR


[P09.16].EVIDENCE OF TRA-1-60 AND TRA-1-81 INVOLVEMENT IN L-SELECTINMEDIATED ADHESION OF THE PORCINE EMBRYO

E Oestrup, V Dantzer*, P Maddox-Hyttel. University of Copenhagen,Denmark

L-selectin expression in the early human embryo is involved in trophoblastadhesion during implantation. The aim of this study was to investigate thepotential role of the L-selectin adhesion-system in the adhesion andplacentation of porcine embryos. Endometrial samples were collectedfrom pregnant gilts and non-pregnant cycling sows at Days 10, 15 and 18post insemination/oestrus. Embryos were collected from pregnant gilts atDays 9, 11 and 15 for expression studies and from Days 10 and 15 forimmunohistochemistry.Expression analysis of L-selectin and its ligands was made using qPCR.Protein localization of L-selectin and the ligands PNAd, PEN5 as well as thepodocalyxin epitopes Tra-1-60 and Tra-1-81 were investigated by immu-nohistochemistry. In pregnant gilts the mRNA expression of L-selectin wasapproximately 14 fold up-regulated at Days 15 and 18 compared to Day 10p.i. No significant changes were seen in the mRNA levels in the cyclic sows.Expression of mRNA for the potential L-selectin ligands, CSPG-2 andpodocalyxin were 6 and 12 times up-regulated in tubular Day 11 blasto-cysts compared to Day 9 blastocysts. Staining for L-selectin was observedin the luminal epithelium of the endometrium. In the embryos, staining forMECA-79 and PEN5 were observed in trophoblast and hypoblast cells atday 15. Tra-1-81 and Tra-1-60 was confined to the epiblast at Day 10 but atDay 15 staining for both epitopes were observed in the trophoblast. Ourresults for the first time demonstrate that key components of the L-selectinadhesion system are present in the porcine uterus and embryo around thetime of initial placentation. Interestingly, the components are expressed ina pattern opposite to that found in man: In the pig, L-selectin is expressedin the endometrium and the ligands in the trophoblast with the reversebeing true in man.Keywords: Porcine, Implantation, L-selectin

[P10.01].EFFECTS OF INSULIN ON MEMBRANE LIPID COMPOSITION OF HUMANSYNCYTIOTROPHOBLAST

M Castro-Parodi*, A Reca, V Dietrich, C Rodrıguez, MC Fernandez-Tome, AEDamiano. Catedra de Biologıa Celular, Depto. de Ciencias Biologicas, Fac-ultad de Farmacia y Bioquımica, Universidad de Buenos Aires, Argentina

Introduction: Altered placental membrane lipid composition in preg-nancy may affect the fetal-maternal exchange. With gestational progress,the composition, structure and functions of these membrane lipid bilayersare modified in order to meet the changing metabolic needs of the growingfetus.Previously, we reported that plasma membranes of syncytiotrophoblast(hST) are unusual in comparison to other cell types. Because of the increaseof sphingomyelin we also informed that preeclamptic hST is more rigidthan normal hST. Since we observed high serum levels of insulin inpreeclamptic women, we hypothesized that insulin may be implicated inthe changes observed in preeclampic hST.The aim of this study was to evaluate if the insulin may alter the lipidcomposition of hST.Methods: Explants from normal term placentas were cultured withdifferent concentrations of insulin during 24 h. The biochemical viability ofthe explants was determined by estimation of b-hCG concentrations in theextracellular medium.Apical (MVM) and basal (BM) membrane vesicles were prepared bydifferential centrifugation.Lipid were extracted by Bligh-Dyer method and quantified by Fiske-Sub-arrow. Cholesterol was determined by enzymatic method.Results: Insulin treatment produced no changes on the total phospholipidconcentration in BM. On the contrary, MVM showed an increase in totallipid content until reaching a constant value at 100 mUI/mL of insulin.There were no changes in the amount of cholesterol in both vesicles.Discussion: Our results suggest that insulin treatment only alter phos-pholipid concentration in MVM having no effect on BM vesicles. Furtherwork is necessary to clarify the molecular mechanisms implicated inthese changes and if they may play a role in the pathogenesis ofpreeclampsia.Keywords: insulin, lipids, syncytiotrophoblast, preeclampsia


[P10.02].PERFUSION OF THE HUMAN PLACENTA WITH FREE HEMOGLOBINMIMICS PREECLAMPSIA IN-VITRO

M Centlow*1, H Schneider3, S Hansson1. 1Department of Obstetrics &Gynecology, Lund University, Sweden, 2Division of Infection Medicine,Lund University, Sweden, 3Department of Obstetrics and Gynecology, InselSpital, University of Bern, Switzerland

Background: We have previously shown fetal hemoglobin (HbF) mRNAexpression to be increased in the preeclamptic placenta and accumulatedin the placenta blood vessels where it damages the feto-placental barrier,causing leakage of free HbF into the maternal blood circulation. Theincreased levels of HbF induce placental gene expression of alpha-1-microglobulin (A1M) and increase maternal plasma levels of A1M. Alpha-1-microglobulin is a heme and radical-scavenger, and has reductase- andanti-oxidative properties.Aims: We used the in vitro dual-placenta perfusion model to evaluate themolecular effects of free HbF on the placenta.Material and Methods: 20 placentas were collected at birth and perfusedin the dual placenta perfusion model with perfusion medium only (n¼6), 2mg/ml free Hb (n¼9) or 30 mM a1m (n¼3). Placental tissue samples werecollected before and after a successful perfusion. Effects on the placentawere analyzed with Illumina whole-genome Beadarrays and the histo-logical structure was evaluated with electron microscopy.Results: Hemoglobin perfusions resulted in increased gene expression ofamongst other toll-like receptor adaptor molecule 1 (fold change (FC)¼8.5,p¼1.3�10-5) and oxidative stress induced growth inhibitor 1 (FC¼10.8,p¼1.9�10-4). A1M perfusions increased gene expression for g-coupledprotein receptor 1 (FC¼11.6, p¼1.6�10-4) and collagen, type IV, alpha 5(FC¼3.6, p¼1.6�10-4).Conclusions: Free Hb is well known to have pro-inflammatory and pro-oxidative properties and interestingly, among the most increased genesseveral oxidative markers are seen. Thus, it is possible that free Hb not onlyoxidizes proteins and lipids but also induce oxidative changes on a geneexpression level. Alpha-1-microglobulin seems to not only function asa scavenger, it may also up-regulate genes related to extra-cellular matrix,indicating that A1M could aid in extra-cellular matrix repair whereoxidative damage has occurred.Keywords: Preeclampsia, Placenta perfusion

[P10.03].ECCENTRIC CORD INSERTION AFFECTS BIRTH WEIGHT IN THECOLLABORATIVE PERINATAL PROJECT (CPP)

CM Salafia2, DP Misra3, AK Charles*1, RK Miller4. 1University of WesternAustralia, Australia, 2Placental Analytics, United States, 3Wayne StateUniversity, United States, 4University of Rochester, United States

Background: Fetal growth is a complex process depending on genetics,uterine environment and placental function. The placenta develops ideallywith a near centrally inserted cord, an eccentric cord is due to non uniformradial growth of the placenta. This is likely to be due to less than idealintrauterine environment. Previous (smaller) studies have shown a vari-able relationship between non central cord insertion and birthweight. Thehypothesis was that there would be an association with an eccentric cordinsertion with a low birthweight.Methods: Linear regression was used with product terms to model inter-actions between predictor variable pairs including cord eccentricity (therelative distance of cord insertion from placental margin), disk eccentricity(the ratio of the larger and smaller chorionic disk diameters), andabnormal disk shape (categorized as ‘‘round/oval’’ v. all other shapes), tofetal and placental weight in the 29475 infants of the NCPP cohort with therequired measures.Results: A more centrally inserted cord was associated with a small butreliable direct (positive) effect on birthweight (¼ 73�23g, , p¼0.001), butno effect on placental weight (p¼0.49). A regression of the three measures(umbilical cord insertion and chorionic disk eccentricities and normal v.abnormal disk shape) revealed that each of the three measures retainedindependent and significant predictive effects on birthweight (cordcentrality ¼ 65�23, disk eccentricity ¼ -114�21, and disk shape -57�16,p¼0.004, p<0.0001 and p<0.0001 respectively).Conclusion: A non central cord insertion is associated with a reduced birthweight in this large cohort. The pathophysiology of this may be due toa suboptimal intrauterine environment leading to non uniform placentalgrowth and affecting the birth weight, but also the less than optimalvascular branching on the chorionic plate may affect the efficiency of theplacenta. The findings support the trophotropism theory of placentaldevelopment.Keywords: Placenta, Birthweight, Growth


[P10.04].IDENTIFICATION OF DOWNSTREAM TARGET GENES OF THE HOMEOBOXGENE DLX3 IN THE HUMAN PLACENTA

A Chui*1,2, B Kalionis1, N Pathirage1,2, SP Brennecke1,2, P Murthi1,2. 1TheRoyal Women’s Hospital, Australia, 2The University of Melbourne, Australia

Introduction: Fetal growth restriction (FGR) is a pregnancy disordercharacterised by birth weight below the 10th percentile for gestational agewith the likely presence of an underlying pathologic process that inhibitsthe expression of the normal intrinsic growth potential. In the humanplacenta, the homeobox gene DLX3 has been shown to be altered in FGR.Our recent work shows that DLX3 is implicated in the regulation oftrophoblast cell differentiation. In this study, the aim was to determine thepathways by which DLX3 regulates trophoblast differentiation.Methods: BeWo cultured cells were treated with DLX3-specific siRNA tosignificantly decrease DLX3 gene expression. cDNA was prepared fromsiRNA treated and control siRNA cells. A PCR-based low density ‘‘Super-array’’ system was used for gene profiling to identify the downstreamtarget genes of DLX3. Candidate genes from the array, which showeddifferential expression between siRNA treated and control siRNA cells,were chosen for further validation in FGR-affected human tissue (n¼25)compared with gestation matched controls (n¼25), by real-time PCR.Results: Decreased expression of DLX3 resulted in significant increased(>50) fold changes in the expression of all the following genes: CEBPB,ESR1, GATA2, GATA3, TNFRS11A, PPARg, SMAD5, SP1, STAT2 and TP53. Ofthese genes, verification in placental tissue affected by FGR was performedfor PPARg and SP1. The mRNA expression of both SP1 (2.81�1.138 controlvs. 29.22�5.0 FGR, P¼0.0000343) and PPARg (4.9�0.56 control vs.55.52�9.49 FGR, P¼0.00097) was significantly increased in FGR-affectedplacental tissue compared with controls.Discussion: This study has identified downstream target genes of DLX3including CEBPB, ESR1, GATA2, GATA3, TNFRS11A, PPARg, SMAD5, SP1, STAT2and TP53. DLX3 may therefore play a key role in many aspects of differ-entiation, since these target genes affect multiple signal transductionpathways.Keywords: Homeobox gene, Trophoblast, Fetal Growth Restriction

[P10.05].X-SHAPED FUSED-FORKED UMBILICAL CORD IN A MONOAMNIOTICTWIN PREGNANCY AS A RARE VARIANT OF CONJOINED TWINNING

S Dekan*, Y Bader, M Stammler-Safar, G Poschalko-Hammerle, D Prayer.Medical University Vienna, Austria

Monomaniotic twinning is rare and accounts for 1 – 2 % of all monocygotictwin gestations, and is associated with high mortality rates of 10% to 20%due to entanglement of the cords or knotting, problems accounting topremature delivery, twin-to-twin-transfusion syndrome and malforma-tions including conjoined twinning. Fusions of umbilical cords are rare, 9out of 10 cases reported in literature occurred in monoamniotic twins,whereas only one case was reported in diamniotic twins. All of these casesshowed y-shaped fused umbilical cords. The overall outcome was poorwith only one case with 2/10 healthy survivors and 8/10 fetal demises.We show the first case of a monochorionic monoamniotic pregnancy witha x-shaped fused umbilical cord. The fusion of the cords was detected ina routinely performed fetal MRI with 6 vessels in the fused part. Atgestational age of 32+2 weeks two girls were delivered by caesareansection. The children were appropriate to gestational age in weight andmaturity and developed normaly.The monoamniotic placenta weighting 384 gramms showed two umbilicalcords inserting centrally at a distance of 0.8 cm, containing three vesselseach. After 1.2 cm the cords Wharton jellies were fused over a distance of6.3 cm, containing 6 vessels. After the separation of the two cords each had3 vessels again. The placental size, weight and villous parenchyme matu-rity matched gestational age.The most popular theory of development of forked umbilical cords is dueto defectional separation during embryonic fission after developmentalday 9/10 when the formation of the amnion is finished, but before day 12,when conjoined twinning would result. Another theory by Spencer is thefusion of two monovular embryos, which could be consistend with themonochorionic diamniotic case and the case presented here.Keywords: umbilical cord, fusion, twinning


[P10.06].PLACENTAL MORPHOMETRY RELATED TO MATERNO-FETAL BLOODFLOW IN PRE-ECLAMPSIA

JF Ducray*1, T Naicker2, J Moodley2. 1Durban University of Technology,South Africa, 2University of Kwa-Zulu Natal, South Africa

Introduction: Adequate maternal, intervillous and fetal blood flow are allnecessary for fetal wellbeing. Compromise to any part of this exchangewould be detrimental to pregnancy outcome. Pre-eclampsia is associatedwith reduced maternal spiral artery flow, resulting in reduced placentalperfusion. This in turn creates an ischaemic environment which may pre-dispose morphological changes in placental villi.Methods: This study utilized morphometric image analysis to examineselected placental features associated with materno-fetal exchange innormotensive (NT) and pre-eclamptic (PE) groups. The features examinedincluded: density of placental villi (expressed as percentage of field areaoccupied by placental tissue); stem vessel carrying capacity (expressed aspercentage of stem villus area occupied by vessel lumina); the relativethickness of the stem arterial walls (expressed as percentage of artery areaoccupied by arterial wall) and the extent of fibrosis associated with villi(expressed as percentage of field area occupied by fibrosis).Results: Density of placental villus arrangement NT:51.89�6.19,PE:64.78�6.93 (P¼155232E-10); carrying capacity of stem villiNT:17.20�11.78, PE:8.67�8.51 (P¼1.19097E-05); relative thickness of stemvilli arterial walls NT:74.08�12.92, PE: 86.85�10.55 (P¼2.04099E-08); andextent of fibrosis NT:0.727�0.310, PE:1.582�0.707 (P¼1.66E-07).Discussion: It is well established that the maternal side of placentalexchange is compromised in pre-eclampsia. However, precisely howplacental development and function is altered as a result is not clear. Onewould expect possible morphological compensation or ischaemic changes.The significant differences between normotensive and pre-eclampticplacentae observed in this study suggest possible fetal maladaptations inresponse to the intervillous ischaemia, compounding the existing maternalcompromise to materno-fetal exchange.Keywords: Pre-eclampsia, materno-fetal exchange, placental villi, micro-scopic placental morphometry

[P10.07].EXPRESSION OF THE RECEPTOR FOR ADVANCED GLYCATION ENDPRODUCTS (RAGE) AND ITS SOLUBLE FORM SRAGE IN PRE-ECLAMPTIC PLACENTAS AND AGE-MATCHED CONTROLS

S. Dekan, Y.-A. Chen, H. Uhrova, G. Poschalko-Hammerle, I. Ellinger*.Medical University Vienna, Austria

Introduction: The receptor for advanced glycation end products (RAGE)binds a variety of ligands. The ligand-RAGE axis contributes to a widespectrum of diseases, including diabetes mellitus or atherothrombosis.Soluble forms of RAGE (sRAGE) may counteract RAGE-mediated patho-genesis by acting as a decoy. Decreased levels of sRAGE may serve asa biomarker of ligand-RAGE axis hyperactivity, but also providing a targetof therapeutic interventions. Severe pre-eclampsia (PE) was found asso-ciated with increased levels of RAGE ligands, but alterations of placentalRAGE expression remain controversial. Levels of RAGE and sRAGEexpression in healthy and pre-eclamptic placentas are unknown, but thisknowledge is required to argue in favour or against a contribution of theligand-RAGE axis to the development of PE. This study is concerned withthe analysis of RAGE and sRAGE expression in healthy human termplacenta, in pre-eclamptic placentas and their age-matched controls.Methods: RAGE/sRAGE expression was investigated by RT-PCR, westernblotting and microscopy in term placentas. RAGE/sRAGE expression inmoderate to severe pre-eclamptic placentas and their age-matchedcontrols was analyzed by immunofluorescence microscopy of paraffin-embedded sections applying RAGE and sRAGE specific primary antibodies.Results: RAGE and sRAGE transcripts were detected in healthy termplacental tissue by RT-PCR. By western blotting, RAGE and sRAGE wasdetected using antibodies directed against either the C- or N-terminus ofRAGE, respectively, but sRAGE was the predominant protein expressed inhealthy term placental lysates. By immunofluorescence microscopy, bothsRAGE and RAGE were localized to STB but also endothelial cells in situ.Finally, the expression of RAGE in relation to sRAGE was significantlyincreased in STB and endothelial cells of PE-derived placentas compared totheir age-matched controls.Discussion: Our data indicate an up-regulation of full-length RAGE in PE-derived placentas and suggest an involvement of the ligand-RAGE axis inthe development/aggravation of PE.Keywords: RAGE, sRAGE, preeclampsia


[P10.09].EFFECT OF ABNORMAL MATERNAL IMMUNE ACTIVATION ONPREGNANCY SUCCESS IN THE RAT

SJ Renaud*, J Quirt, SK Macdonald-Goodfellow, CH Graham. Queen’sUniversity, Canada

Introduction: Aberrant activation of the maternal immune system is animportant aspect of pregnancy-related diseases including pre-eclampsia,fetal growth restriction, and spontaneous abortion. Administration oflipopolysaccharide (LPS) has been used in rodents to model the afore-mentioned pregnancy-related diseases. However, the mechanisms bywhich LPS exerts negative effects on pregnancy are not fully understood.Therefore, we determined the outcomes of LPS administration on preg-nancy success in the rat, in relation to changes in placental blood flow andlevels of pro-inflammatory and thrombotic molecules.Methods and Results: Administration of LPS (50, 75, or 100 mg/kg) to Wistarrats on gestational day 14.5 resulted in fetal loss in a dose-dependentmanner; furthermore, surviving fetuses were significantly growthrestricted. These outcomes correlated with elevated levels of tumournecrosis factor-a (TNF) in maternal blood, as well as increased expression ofcyclooxygenase-2 and inducible nitric oxide synthase (iNOS) in decidualtissues. Placental blood flow was impaired within 2 h of LPS administrationas determined by Doppler ultrasound and there was hemorrhage in thedecidua and the fetal-maternal interface. Evidence of disseminated intra-vascular coagulation was present within 1 h of LPS injection as determinedby thromboelastography, concomitant with increased levels of plasminogenactivator inhibitor-1 in maternal blood. LPS also caused placental oxidativestress as determined by immunoblotting for 4-hydroxy nonenal. Injection ofinterleukin-10 prior to LPS, but not the iNOS inhibitor aminoguanidine,improved fetal outcomes, concomitant with decreased TNF levels.Conclusion: We conclude that abnormal maternal immune activationnegatively affects rat placental development and that LPS administrationprovides a useful model to study pathological pregnancies.Funding source: Canadian Institutes of Health Research and Heart andStroke Foundation of Canada.Keywords: inflammation, pre-eclampsia, IUGR, spontaneous abortion

[P10.10].THE DIFFERENT REGULATION OF HO-1 AND INOS IN PLACENTA OFPREECLAMPSIA

Jongyun Hwang*, Jiyeon Lee, Youngmyeong Kim. Kangwon NationalUniversity, Republic of Korea

Background: Heme Oxygenase (HO) and nitric oxide synthase (NOS) havesimilarities in some aspects.HO and NOS play significant role in placentation, placental angiogenensisand antioxidant protection from oxidative stress in normal pregnancy.Some researcher suggested that HO and NOS may be regulated by mutualisoform enzymes.However, the exact comprehension for the compensatory regulationbetween two enzymes was deficient.Preeclampsia (PET) is a hypertensive disorder in pregnancy. It is hypoth-esized that the cause of PET is disturbance of spiral artery modification oftrophoblast in the early placentation. The placenta hypoxia results in theinitiation of maternal inflammatory cascade with endothelial dysfunctionin PET.We hypothesized that the deficiencies of HO, HO-catalytic products, NOSand NOS-catalytic products may result in PET and there is a reciprocalcompensatory system in two systems.Objects: The aim of this study was to identify the gene expression of HO-1and iNOS in placenta of PET and to evaluate the potential reciprocalcompensatory regulation between two systems.Methods: In this study, we designed Case-control study including fourteenwomen with PET. The placenta tissue samples were obtained from PETpatients and normal pregnancy. We quantified gene expression of HO-1and iNOS on placenta by RT-PCR, real time PCR.Results: In this study, it demonstrates that a significant down-regulationof HO-1 gene expression in placenta when compared to normal placenta.However, we observed a significant up-regulation of iNOS gene expressionin placenta when compared to normal placenta.Conclusion: The present study demonstrates that there are differentregulation to preeclampsia in two enzyme systems; the down-regulationof HO-1 gene expression and up- regulation of NOS gene expression. Wesuggest that HO-1 and iNOS system may be regulated by mutual geneexpression between two enzymes in placenta.Keywords: Heme oxygenase, nitric oxide synthase, preeclampsia, placenta


[P10.11].THE INHIBITORY ROLE OF MCL-1 ON THE INDUCTION OF AUTOPHAGYIN THE HUMAN PLACENTA

M Kalkat*1,2, J Garcia1, J Ray1,2, I Caniggia1,2. 1Samuel Lunenfeld ResearchInstitute, Mount Sinai Hospital, Canada, 2University of Toronto, Canada

Introduction: Autophagy has recently been recognized both as an adap-tive mechanism to cellular stress and an alternative cell death pathway.Placental development requires a balance between proliferation anddeath; however the relative contribution of autophagy in normal andpathological pregnancies remains elusive. Preeclampsia (PE), a complica-tion of pregnancy, is characterized by oxidative stress and increasedtrophoblast cell death. Biochemical analysis implicated an essentialautophagy inducer, Beclin-1, to interact with some Bcl-2 proteins. We havepreviously found Myeloid Cell Leukemia Factor-1 (Mcl-1), a Bcl-2 familymember, to guide trophoblast cell fate following changes in oxygenation.Herein we sought to examine the role of Mcl-1 on autophagy in normaland pathological placentae.Methods: First trimester (4-15 weeks; n¼26), age-matched (AMC; n¼8)and term controls (TC; n¼9) and preeclamptic placentae with Hemolysis,Elevated Liver enzymes and Low Platelets (PE-HELLP; n¼14) were used.Immunoblotting was performed to determine protein expression of Mcl-1,Beclin-1 and the autophagosome marker LC3. Immunoprecipitationstudies were conducted to test Mcl-1/Beclin-1 association. To examine therole of Mcl-1 in trophoblast autophagy, Mcl-1 was overexpressed inchoriocarcinoma JEG3 cells. JEG3 cells were treated with sodiumnitroprusside (SNP; 5 mM), a nitric oxide donor to mimic oxidative stress.Results: Immunoblotting revealed increased Beclin-1 and LC3 expressionat 12-15 weeks gestation, correlating to decreased Mcl-1 protein levels.Overexpression of Mcl-1 in JEG3 cells decreased LC3 expression. Exposureof JEG3 cells to SNP resulted in decreased Mcl-1 followed by increased LC3expression. Immunoprecipitation studies verified Beclin-1/Mcl-1 interac-tion. Increased expression of LC3 was observed in PE/HELLP placentae andit was associated with decreased Mcl-1 levels.Discussion: Herein we demonstrate that autophagy plays a role duringplacental development and that Mcl-1 is a direct inhibitor of autophagy introphoblast cells. Increased autophagy in preeclampsia may be due todecreased Mcl-1 expression (Supported by CIHR and OWH/IGH).Keywords: Preeclampsia, Autophagy, Oxidative Stress, Bcl-2 Family

[P10.13].THIRD TRIMESTER STILLBIRTHS: CORRELATIVE PLACENTALPATHOLOGY AND NEUROPATHOLOGY

KTE Chang*1, P Shannon2, G Machin2, J Kingdom2, S Keating2. 1The Hospitalfor Sick Children, Canada, 2Mount Sinai Hospital, Canada

Introduction: Placental pathology often identifies the reason for stillbirth,providing important information for future obstetrical management. Inthis study, we aimed to describe the neuropathology of third trimesterstillborn fetuses in relation to placental pathology and selected autopsyfindings and to identify specific placental lesions which correlate withneuropathological findings.Methods: This was a retrospective review of third trimester stillbirthautopsies performed at Mount Sinai Hospital. Fetuses with congenitalmalformations and abnormal karyotypes were excluded. Placental path-ological findings were studied in the categories of maternal vascular, fetalvascular, inflammatory and cord pathology. Meconium staining, thepresence of increased nucleated red blood cells and thymic stress changeswere also studied. Neuropathological findings were categorized as follows:(i) neuronal injury (grade 0–absent, 1–early injury, 2–pontosubicularnecrosis, 3–widespread neuronal injury), (ii) gliosis (0–absent, 1–present),and (iii) white matter injury (0–absent, 1–white matter edema, 2–swollenaxons and/or necrosis). The Fisher’s exact test was used to identifystatistically significant associations.Results: 37 cases of stillbirth were studied. The brains in 22 cases showedneuronal injury grades 2 and 3. 14 cases had gliosis. Two cases showedgrade 2 white matter injury. There was significant correlation of acutechorioamnionitis, meconium staining and thymic stress reaction withneuronal injury of grades 2 or 3, and with gliosis. Acute chorioamnionitisalone and placental inflammation overall both showed significant corre-lation with neuronal injury grades 2 and 3. There was no correlation of theplacental parameters with grade 2 white matter injury.Discussion: Grey matter injury and gliosis were the most commonneuropathological findings in these third trimester stillbirths. Whitematter damage and germinal matrix hemorrhage were rare in this pop-ulation. Of the categories of placental pathology examined, inflammatorylesions showed the strongest correlation with neuronal injury of the fetalbrain.Keywords: Stillbirth, Neuropathology, Acute chorioamnionitis


[P10.14].FREQUENCY OF PATHOLOGICAL CHANGES IN MORPHOLOGICALLYNORMAL PERINATAL DEATHS

D.N. Kenwright*, N. J. Sidek, S. Jaffar, J. Zuccollo. University of Otago,Wellington, New Zealand

Introduction: Placental pathology frequently contributes to perinataldeath. In this study we examined a 12 year cohort of perinatal deaths inWellington, New Zealand to determine which maternal and placentalpathologies contribute to the deaths and the relationship betweenplacental changes and maternal disease.Method: 725 reports of postmortems performed at Wellington PublicHospital on perinatal deaths from 20 weeks gestation to term wereexamined and the placental and maternal pathologies recorded inmorphologically normal babies. These data were then analysed using t-test, Chi-square and Fisher test to determine the odds ratio (OR) and theirsignificance.Results: From 725 morphologically normal babies, 691 placentas wereexamined and placental pathology was found in 447 placentas. 139 preg-nancies were complicated by maternal disease. Frequency of eachplacental pathology are as below:

Placental pathology Frequency Gestational age at which frequency
(%) p eaks
Chorioamnionitis
18.5 2 0-23 wk Funisitis 10.1 2 0-23 wk Abruption 10.1 2 2-23 wk Infarction 18.4 2 2-26 wk Significant/massive
perivillousfibrin deposition

9.1 N
o specific age
Villitis of unknown aetiology
8.7 3 8-42 wk Lymphohistiocytic
intervillositis
2.3 N o specific age
Decidual vasculopathy
2.3 N o specific age Fetal/maternal haemorrhage 2.3 N o specific age Rupture of fetal vessel 0.3 N o specific age Subchorial haemorrhage 2.9 N o specific age Fetal thrombotic
vasculopathy
5.6 N o specific age
Villous hypoplasia
6.2 2 2-26 wk Villous dysmaturity 4.8 3 7-42 wk
Statistically significant correlation was found between maternal diabetesand villous dysmaturity (OR: 5.91, p<0.05). Pregnancies complicated bymaternal hypertension was found to correlate with chorioamnionitis(OR:3.127, p<0.05) and funisitis (OR: 2.76, p<0.05). Pregnancies compli-cated by PET have increased chorioamnionitis (OR:9.3, p<0.000), funisitis(OR:2.81, p¼0.05), decidual vasculopathy (OR:21.1, p<0.000), infarction(OR:2.21, p<0.05), and villous hypoplasia (OR:4.0, p<0.05). HELLPsyndrome correlates with infarction (6.1, p<0.05) and villous hypoplasia(OR:12.1, p<0.05). IUGR correlates with maternal hypertension (OR:7.1,p<0.000), PET (OR:32.3, p<0.000), HELLP (OR:15.3, p<0.01).Conclusion: This is the first comprehensive review of placental pathology inNew Zealand. The high rate of placental morphologic abnormality found inmorphologically normal infants shows the importance of placental exami-nation in perinatal post mortems. Our data reflects previous findings in thathigh rates of chorioamnionitis and abruption are found in very early pretermdeliveries. The histological findings in the placenta with maternal diseasesare similar to other reports, with the exception of maternal hypertensionand chorioamnionitis/funisitis which was a surprise correlation.Keywords: placenta, pathology, perinatal, death

[P10.15].INSULIN-INDUCED VASODILATION IS REDUCED IN HUMAN UMBILICALARTERIES FROM GESTATIONAL DIABETES

BJ Krause*, MJ Jo, P Casanello, L Sobrevia. Cellular and Molecular Physi-ology Laboratory (CMPL) and Perinatology Research Laboratory (PRL),Department of Obstetrics and Gynecology, Medical Research Centre (CIM),School of Medicine, Pontificia U, Chile

Insulin increases the activity of nitric oxide synthases (NOS) and acts asvasodilator in several vascular beds. Gestational diabetes (GD) is asso-ciated with elevated fetal plasma insulin level compared to normalpregnancies, but fetuses from GD could exhibit either insulin resistanceand low birth weight, or be responsive to insulin with high birth weight.In addition, placental blood flow correlates with birth weight in GD. Westudied insulin vasoactive effect and the potential NOS role in umbilicalarteries from normal and GD pregnancies. METHODS. Umbilical arteriesfrom normal and GD pregnancies were dissected and vessel rings weremounted on a wire-myograph. Isometric force in response to insulin(0.001-10 nM) in the presence or absence of the NOS inhibitor N-nitro-L-arginine (L-NA, 100 mM) was measured in KCl (37.5 mM) pre-contractedvessel rings. Responses were expressed as a percentage of relaxationrelative to corresponding maximal effects of KCl. RESULTS. Insulininduced vasodilatation (18.2�1.3%) in a concentration-dependentmanner (half-maximal effect¼10.8�0.2 nM) in vessels from normalpregnancies (n¼4), a response blocked by L-NA. However, insulininduced either vasodilatation, which was partially inhibited (w55%) byL-NA, or vasoconstriction in GD (n¼6), an effect that correlated (r2¼0.9)with fetal weight. CONCLUSIONS. Human umbilical artery dilatationinduced by insulin was dependent or partially dependent on NO innormal or GD pregnancies, respectively. Variability of the vasoactiveresponse to insulin in umbilical arteries from GD could in part explainaltered placental blood flow in fetuses small or large for the gestationalage.Supported by FONDECYT 1070865/1080534. B.J.K. holds a CONICYT-PhDfellowship.Keywords: insulin, vasodilation, umbilical, vessels

[P10.17].UP-REGULATION OF THE IGF2 GENE EXPRESSION BY HYPOXIA INPREECLAMPSIA AND INTRAUTERINE GROWTH RESTRICTIONPLACENTAS

C Louet*1,4, S Barbaux1, J Tost2, C Buffat3, D Vaiman1, H Jammes1,4. 1InstitutCochin, France, 2Centre National de Genotypage, France, 3Hopital LaConception, France, 4INRA, France

The paternally imprinted IGF2 gene encodes the Insulin-like growthfactor 2 (IGF2), a key regulator of placental development whosedysfunctions are implicated in human pregnancy pathologies such aspreeclampsia and/or intrauterine growth restriction. We first performeda thorough update of the structure of IGF2 transcripts and promoters inhuman using extensive in silico data. We found an increase of IGF2 geneexpression in all pathological placentas versus normal placentas. Ana-lysing the methylation status at the IGF2/H19 locus (56 CpGs) by pyro-sequencing, a tissue-specific hypomethylation of the IGF2 DMR2 wasfound in human placentas. Nevertheless, the imprinted status of IGF2 andH19 was conserved in all placental diseases. We investigated the use ofthe five IGF2 promoters in order to elucidate the IGF2 gene up-regulation.It is mainly caused by the use of P0 promoter, correlated to the inductionof YY1, a human P0 promoter specific transcription factor implied in theregulation of imprinted genes. Moreover hypoxia up-regulates theexpression of the IGF2 transcript P0 in a human choriocarcinoma cellmodel. In the light of these results we hypothesise that, in addition to itsprimary imprinting regulation, the complex transcriptional regulation ofthe IGF2 gene permits an adaptation to the hypoxic environment presentin placental diseases.Keywords: IGF2, hypoxia, placental pathologies


[P10.18].PLACENTAL BIGLYCAN EXPRESSION IS DECREASED IN HUMANIDIOPATHIC FETAL GROWTH RESTRICTION

P Murthi*1,2, FA F1,2, G Rajaraman1,2, NA Pathirage1,2, V Ignjatovic3,4, JMSaid1,2. 1University of Melbourne, Australia, 2Royal Women’s Hospital,Australia, 3Murdoch Children’s Research Institute, Australia, 4The RoyalChildren’s Hospital, Australia

Introduction: Fetal growth restriction (FGR) is a leading cause ofprenatal morbidity and mortality. The majority of FGR cases are idio-pathic and are associated with placental insufficiency, which can resultfrom placental thrombosis. Evidence suggests that Dermatan Sulfate (DS)is an important anticoagulant in placentae of uncomplicated pregnan-cies. This study hypothesised that the expression of biglycan proteo-glycan, a source of DS, is decreased in idiopathic FGR placentaecompared with placentae from uncomplicated pregnancies. This studyaimed to determine biglycan mRNA, protein expression and spatialdistribution in idiopathic FGR placentae compared with the placentaefrom gestation-matched controls.Methods: This study investigated 26 placentae from idiopathic FGR-affected pregnancies and 27 placentae from uncomplicated, gestation-matched pregnancies (27 to 40 weeks gestation). Inclusion criteria for theidiopathic FGR group included a birth weight of less than the 10th

percentile and at least two of the following; abnormal umbilical arteryDoppler velocimetry, oligohydramnios or asymmetric fetal growth.Biglycan mRNA expression, protein expression and spatial distributiondetermined using real-time PCR, immunoblotting and immunohisto-chemistry, respectively.Results: Mean biglycan mRNA expression was significantly decreased inFGR placentae compared with control placentae (2.87�1.93 (n¼26) vs4.48�2.40, (n¼27) p¼0.01). FGR placentae demonstrated significantlydecreased mean biglycan protein expression compared with controlplacentae (23.9�7.7 vs, 59.3�10.7, n¼12, p<0.05). Biglycan immunoreac-tivity was detected in endothelial cells and smooth muscle cells lining thefetal capillaries. Semi-quantitative analyses demonstrated a significantdecrease in immunoreactive biglycan in FGR placentae compared withcontrol placentae (51.1�19.3 vs, 500.7�223, n¼6, p<0.001)Discussion: This is the first study to demonstrate the association betweendecreased biglycan expression and idiopathic FGR placentae. Reducedbiglycan expression may contribute to placental thrombosis within the fetalvasculature, and may contribute to the pathogenesis of idiopathic FGR.Keywords: fetal growth restriction, placenta, proteoglycans, thrombosis

[P10.19].VITAMIN C BUT NOT VITAMIN E INCREASES THE EXPRESSION OF PP13AND BETA-hCG IN FORSKOLIN STIMULATED BeWo CELLS

K Orendi*1, M Gauster1, G Moser1, H Meiri2, B Huppertz1. 1Institute of CellBiology, Histology & Embryology, Medical University Graz, Austria, Austria,2Diagnostic Technologies Ltd, Yokneam, Israel, Israel

Objectives: Maternal serum concentrations of placental protein 13 (PP13)have been shown to be altered in first trimester pregnant women subse-quently developing preeclampsia compared to controls. Vitamins C and Eare putative therapeutics for prophylactic treatment of preeclampsia. Herewe used the choriocarcinoma cell line BeWo as surrogate for primarytrophoblast and investigated the influence of vitamins C and E on theexpression of PP13 and beta-hCG in these cells.Methods: BeWo cells were cultured for 48h with increasing concentra-tions of vitamin C (30 to 200 mM) and the vitamin E derivative Trolox (10 to100 mM) in presence or absence of 20 mM forskolin. Culture supernatantsand cell lysates were collected to determine PP13 and beta-hCG expressionand release by Delfia assays. Cell viability was estimated by LDH detectionin supernatant. Immunofluorescence was performed using antibodiesagainst beta-hCG as a marker for differentiation and E-cadherin to visu-alize cell fusion.Results: Without forskolin stimulation vitamins C and E did not show anyeffects on the expression of PP13 in cell lysates. After forskolin stimulation,PP13 and beta-hCG concentrations in cell lysates significantly increasedwith increasing vitamin C concentrations in a dose-dependent manner.Viability did not show any changes in any of the conditions. Morphologicalanalysis of immunofluorescent staining with beta-hCG indicated a higherrate of cell differentiation after vitamin C supplementation. Addition ofvitamin E did not show to have an effect on the expression of PP13 but ledto an increased expression of beta-hCG.Conclusions: Vitamin C but not vitamin E has a positive effect on theexpression of PP13 and beta-hCG in differentiating BeWo cells compared tocontrols. It needs to be elucidated whether a similar effect is present inprimary cells and in vivo as well.Keywords: Preeclampsia, Vitamin C+E, Placental Protein 13/PP13


[P10.20].TGIF EXPRESSION AND SIGNALLING IN HUMAN VILLOUS ANDEXTRAVILLOUS TROPHOBLASTS

NA Pathirage*1,2, B Kalionis1, SP Brennecke1,2, P Murthi1,2. 1Department ofPerinatal Medicine, Pregnancy Research Centre, Royal Women’s Hospital,Victoria, Australia, 2Department of Obstetrics and Gynaecology, TheUniversity of Melbourne, Victoria, Australia

Introduction: Abnormal homeobox gene expression has previously beenassociated with the clinically significant disorder fetal growth restriction(FGR) (1). Targeted disruption of transforming growth beta-induced factor(TGIF), a member of the TALE (three-amino-acid loop extension) super-family of homeobox genes has been shown to result in placentaldysfunction in the mouse (2). One aim of this study was to determine theexpression patterns and expression levels of TGIF in FGR affected placentacompared with gestation matched controls. The other aim was to identifygenes/regulatory networks modulated by TGIF in human trophoblast cells.Methods: To determine the spatial and temporal expression of TGIFimmunohistochemistry was carried out on first trimester, normal term andFGR affected placental sections. Relative TGIF mRNA expression of FGRplacentae and gestation age matched normal placenta was determinedusing real-time PCR. Western blotting was conducted to assess TGIFprotein expression. Signalling pathways regulated by TGIF were investi-gated by altering TGIF expression in extravillous trophoblast and villoustrophoblast derived cell lines JEG3 and BeWo respectively and using siRNAand over-expression plasmids followed by PCR-array analysis to determinethe transcriptional profiles.Results: Immunohistochemistry localised TGIF protein expression inresidual cytotrophoblast cells, syncytiotrophoblast cells, microvascularendothelial cells and stromal cells. TGIF mRNA levels were significantly upregulated in FGR compared with normal placentae [1.29�0.06 (n¼25) FGRv. 0.78�0.04 (n¼25) normal, P<0.001]. Similarly, TGIF protein expressionwas significantly increased [3970 � 1101 n¼6) FGR v. 2323�644 n¼6)normal, (P<0.05)]. PCR array analysis revealed differentially regulatedgenes from several biological pathways including TGF-b /BMP, Wnt andMAPK signalling pathways. Furthermore, a subsets of genes was identifiedthat were unique to BeWo and JEG3 cells, suggesting that TGIF may regulatedifferent signalling pathways in villous and extravillous trophoblasts.Conclusion: We conclude that TGIF and its downstream target genes maybe important for trophoblast function and may be a contributing factor tothe developmental abnormalities seen in the FGR affected placentae.Keywords: Fetal Growth Restriction, Trophoblasts, Homeobox genes,Transforming growth beta-induced factor (TGIF)

[P10.21].ANALYSIS OF GESTATIONAL CHORIOCARCINOMA ORIGIN AT THE DNALEVEL

V. Repiska, L. Danihel, V. Sisovsky, L. Danisovic, S. Polak*, D. Bohmer.Comenius University, Faculty of Medicine, Slovakia

Gestational trophoblastic disease (GTD) is a diverse group of trophoblastlesions with specific pathogenesis, morphological and clinical features.Gestational choriocarcinoma is a highly malignant tumor derived from thecells of trophoblast. It may arise from mola hydatidosa, after misscarriageor normal delivery, as well as after ectopic pregnancy. Its incidence ishighest in Asia, Africa and Latin America (1:500–1000 deliveries), inEurope, USA and Australia is lower (1:20000–40000 deliveries). Chorio-carcinoma may grow exophytic way in the uterine cavity or endophyticinside the uterus wall. It has grey-white colour with variable sized depositsof bleeding and necrosis. The microscopic picture shows heterogeneouspopulation of trophoblast cells which infiltrate and destroy tissue ofuterus. Chorionic villi are not present. Moreover, extrauterine choriocar-cinomas and very rarely also in placenta tissue, were described. Immu-nohistochemistry proved high expression of hCG, while presence of HPL inthe cells of trophoblast is rare. DNA analysis enables to distinguishparticular forms of GTD and unambiguously prove the origin of nuclearDNA. Currently it is unique possibility for identification of homozygous

and heterozygous forms of complete hydatidiform mole (CHM), as well asto prove origin of gestational choriocarcinoma.DNA analysis of 10 microscopically proven gestational choriocarcinomas(Fig. 1) appeared after CHM was done. DNA obtained from tumor cells andperipheral blood lymphocytes of patients and progenitors was isolated byQIAamp� DNA Blood Mini Kit. For DNA analysis method of PCR amplifi-cation of particular VNTR sequences (ApoB, Col2A a MCT 118) was used.In 9 cases DNA analysis proved origin of gestational choriocarcinoma fromheterozygous CHM (Fig. 2). Malignant transformation of homozygous CHMto choriocarcinoma happens only in 1 case. Obtained results show thatdiagnostics based only on the microscopic observations is insufficient andutilization of sophisticated methods of DNA analysis bring better perspec-tives in diagnostics of gestational choriocarcinomas after CHM pregnancy.

Fig. 1. Gestational choriocarcinoma (Hematoxylin – Eosin, magnification 140x)

Fig. 2. DNA analysis of gestational choriocarcinoma (1 – heterozygous CHM,2 – progenitor, 3 – choriocarcinoma from heterozygous CHM, 4 – patient)

Keywords: Gestational trophoblastic disease, choriocarcinoma, DNAanalysis, VNTR


[P10.22].PLACENTAL SYNDECAN-1 EXPRESSION IS SIGNIFICANTLY REDUCED INHUMAN IDIOPATHIC FETAL GROWTH RESTRICTION

G Rajaraman*1,2, P Murthi1,2, J Said1,2, V Ignjatovic3,4, PT Monagle3,4, SPBrennecke1,2. 1University of Melbourne, Dept of obstetrics and Gynae-cology, Australia, 2Royal Women’s Hospital, Australia, 3Murdoch Children’sResearch Institute, Australia, 4Royal Children’s Hospital, Australia,5University of Melbourne Dept of Paediatrics, Australia

Introduction: Fetal growth restriction (FGR) is a clinically significant preg-nancy disorder in which the fetus fails to grow to its full potential in utero.70% of FGR pregnancies are idiopathic and associated with placentalthrombosis. Syndecan-1 is a proteoglycan containing the heparan sulfateglycosaminoglycan, which binds growth factors and antithrombin. Previousstudies have shown that placental syndecan-1 expression is restricted to theapical surface of villous syncytiotrophoblasts, suggesting its role in anti-coagulation of the intervillous space. However, the potential role of placentalsyndecan-1 in idiopathic FGR is unknown. This study investigated syndecan-1 expression in idiopathic FGR placentae compared with control placentae.Methods: Human placentae from pregnancies complicated by idiopathicFGR (n¼28) and gestation-matched control pregnancies (n¼28), wereselected according to strict clinical diagnostic criteria. Placentae of 27 to 40weeks gestation were obtained. Immunohistochemistry, western immuno-blotting and real-time PCR were employed for analysis of syndecan-1 proteinand mRNA expression, respectively.Results: Semi-quantitative analyses of immunohistochemistry showedsignificantly reduced syndecan-1 protein expression in the villous syncy-tiotrophoblasts of idiopathic FGR placentae compared with controls(141.7�53.5 versus 362.5�162.1, n¼6, t-test, p<0.01). Syndecan-1 mRNAexpression relative to the housekeeping gene GAPDH, was significantlyreduced in idiopathic FGR placentae compared with controls (0.07�0.02versus 4.48�0.91, n¼28, t-test, p<0.001). Immunoblotting using a mousemonoclonal antibody, demonstrated significantly reduced syndecan-1protein expression in idiopathic FGR placentae compared with controls(55.1�12.7 versus 132.3�45.7, n¼6, t-test, p<0.05).Conclusion: This is the first study to demonstrate a significant decrease inplacental syndecan-1 mRNA and protein expression in idiopathic FGR,suggesting a potentially important role of syndecan-1 in the aetiology ofplacental thrombosis in human FGR.Keywords: Plaenta, Fetal growth restriction, Syndecan-1, Thrombosis

[P10.23].THE ABILITY OF ULTRASOUND TO DETECT PLACENTAL PATHOLOGY

M Robertson*1,2, E Amyes1, D.A Ellwood1,2, J Dahlstrom1,3. 1AustralianNational University, Australia, 2Fetal Medicine Unit, The Canberra Hospital,Australia, 3Anatomical Pathology, The Canberra Hospital, Australia

The placenta is generally under appreciated in the examination andmonitoring of fetal health and well-being. Many problems during theantenatal period that lead to perinatal morbidity and mortality can beattributed to placental insufficiency. The aim of this study was to examinethe placenta in utero using ultrasound and compare the findings withpathological features of the delivered placenta. Nineteen pregnant womenwhose antenatal ultrasound had demonstrated one or more intraplacentallesions together with evidence of IUGR or a previous history of IUGR orperinatal death where recruited. Placental ultrasound was performedbefore delivery. Measurements were taken to determine placental featuresincluding position and echogenicity of lesions. Following birth, placentaswere examined macroscopically and microscopically for evidence of cor-responding pathology. From the study, two groups were ascertained. Thefirst group were placentas with very small diameters but increasedplacental thickness that we have termed ‘‘cup-cake’’. The second groupwere placentas that had hypoechoic lesions that were later determined tobe intervillous thrombi. From the small number of participants recruited tothe study, abnormalities of placental size and shape as well as discretelesions from a number of placentas were identified on ultrasound. Thesewere later confirmed at macroscopic and microscopic analysis indicatinga role for ultrasound in detecting of placental pathology.Keywords: Ultrasound, Placental pathology

[P10.24].METHODOLOGIC ISSUES IN THE STUDY OF THE RELATIONSHIPBETWEEN INFECTION AND PRETERM BIRTH

CM Salafia*1,2, DP Misra3, J Miles4. 1Institute for Basic Research, UnitedStates, 2Placental Analytics LLC, United States, 3Wayne State University,United States, 4Rand Corporation, United States

Goal: To determine the structure of the relationships of the histologyscores for acute intraamniotic infection collected in the CollaborativePerinatal Project (CPP).Materials and Methods: 44,427 subjects of the CPP had completehistology scores available for the 9 measures related to acute intraamnioticinfection (neutrophil infiltrates in umbilical cord, amnion of membranesand chorionic plate, decidua, chorionic plate and fetal chorionic vessels).Confirmatory factor analysis was used to determine the relationshipsamong the different markers of maternal inflammatory responses (inamnion, chorion and decidua) and fetal inflammatory responses (in cordand chorionic vessels). The confirmatory structure included both consid-erations of ‘‘whose’’ infiltrates (maternal versus fetal) and tissue sites inwhich they were assessed (assuming that measures made from the sametissue sample would be more highly correlated that those made fromdifferent samples of the same placenta).Results: A single CFA model could not be developed across all CPP sites,indicating that the above assumptions that underlaid our CFA structurewere not valid. A well-fit CFA model conforming to our assumed structureof relationships was developed from the Boston site (N¼10,803). The factorloadings were then applied to the histology scores from the other CPP sites.The scores for the latent variables (maternal and fetal inflammatoryresponses) were compared across sites. Factor loadings, intercorrelationsof maternal and fetal inflammatory scores and the signs of factor loadingswere inconsistent across sites.Conclusion: Histopathology scores of neutrophil infiltrates performed bydifferent observers do not have the same interrelationships and, byextension, the latent variables they are supposed to reflect (i.e., themeasured maternal and/or fetal inflammatory responses) may not beequivalent. The lack of measurement invariance renders the use of scoringneutrophil infiltrates in placental tissues as indicators of the underlyingprocesses of maternal and fetal inflammatory responses problematic.Keywords: acute infection, neutrophils, structural equation modeling,Collaborative Perinatal Project


[P10.25].MODEL FOR OXYGEN TRANSPORT IN THE HUMAN PLACENTA

JS Gill*1, MP Woods1, CM Salafia2,3, DD Vvendensky1. 1Imperial College,United Kingdom, 2Placental Analytics LLC, United States, 3Institute forBasic Research, United States

Goal/Background: The mature placenta is a complex vascular networkextending ultimately to the capillaries of the terminal villi, site of alloxygen and nutrient exchange between the mother and fetus. Respiratorytransfer across the placenta to the fetus occurs in three steps: (i) maternalblood bathes the chorionic villi in oxygen, (ii) oxygen permeates the villussurface and diffuses into fetal capillaries, and (iii) oxygen is transported tothe fetus via the bloodstream.Materials/Methods: Step (ii) has been modelled by the diffusion ofoxygen from the villous membrane and the fetal capillaries. Thestationary concentration c(x,t) of oxygen within each of the villi is thesolution of the two-dimensional Laplace’s equation, Dc ¼ 0, with a fixedconcentration cv at the villous surface and a Robin boundary condition atthe capillary boundary: Dvc/vn ¼ Kc, where v/vn is outward normalderivative, D is the oxygen diffusion constant and K is the permeability ofthe capillary. These equations are solved in regions [panels (d,e,f)]determined by the villus and capillary boundaries obtained from digitizedimages [panels (a,b,c)].Results: The solution for the oxygen concentration determines thediffusive current of oxygen across the capillaries. Many factors areexpected to influence this current, including the numbers and shapes ofvilli and capillaries. Our initial analysis findicates that (c) yields thelargest flux per unit perimeter length and area of the villi, followed by (b)and (a).Conclusions: The geometrical shapes and spatial distributions of the villi andcapillaries are important placental characteristics for the transport of oxygento the fetus. Once the main factors that determine oxygen transport havebeen identified, this approach, applied to digitized placental slides that allowanalysis of many hundreds of villi per slide (and multiple slides per placenta)should provide a quantitative basis for measuring placental oxygen fluxes.

Keywords: chorionic plate, placental growth, fourier analysis

[P10.26].CENTRALITY OF THE UMBILICAL CORD INSERTION IN A HUMANPLACENTA INFLUENCES THE BIRTH WEIGHT

M Yampolsky1, O Shlakter1, CM Salafia*2,3, DH Mandel3. 1University ofToronto, Canada, 2Institute for Basic Research, United States, 3PlacentalAnalytics LLC, United States

Background: We hypothesize that trophotropism, considered to underlieeccentricity of umbilical cord insertion, results in a deformed placenta, lessfunctionallyefficient, and that the more eccentric the cord insertion, the lessefficient the placenta.Materials and Methods: The model is based on a random fractal growthprocess (DLA). By placing the initial seed asymmetrically, the modelproduces placental vascular trees with a non-centrally placed umbilicalinsertion point, whereas the overall shape of the tree remains round tooval. To test this hypothesis, we calculated a measure of roundness bytaking a mean radial distance to the perimeter. The sector was rotated in2.5� increments to produce 24 radial measurements; the mean squaredeviation swas calculated for both real and model placentas. In thephotographs of the UNC placentas the surface vascular branches weretraced and, for each pixel in the chorionic surface, the minimal distance toa traced vessel was calculated. The resulting number is dimensionless(relative chorionic vascular distance D). A lower value means a betterpenetration of the chorionic surface by the blood vessels.Results: For a round placenta, the value of s is zero, the correlation of thevalue of s with the cord displacement variable in the UNC dataset is only 0.04.Thus, non-centrality of the cord insertion produces little effect on the shape. Around placenta with an asymmetrically placed umbilical cord has a signifi-cantly lower vascular penetration, and hence reduced metabolic efficiency.Conclusion: ‘‘Trophotropism’’, the directional growth of the placenta dueto variations in the intrauterine environment, is considered the mostcommon basis for asymmetrical cord insertions. Our data suggest thateven relatively ‘‘mild’’ eccentricity is associated with abnormal develop-ment of chorionic surface vessels. ‘‘Compensation’’ for a problematicintrauterine environment is incomplete, and does not restore the placentato an ‘‘optimally transporting’’ structure.

Figure 1. Top row: the graph of the function r(q) for a round disk with corddisplacement 5. Bottom row: two placental perimeters (P marked in blue, angularradius in red), with the umbilical insertion point placed at the origin.

Keywords: birth weight, Umbilical cord, placental growth, placentalfunction


[P10.27].RELIABILITY OF AUTOMATED NEUTROPHIL QUANTITATION INDIGITIZED H&E STAINED SLIDES: PILOT ANALYSIS OF CORRELATIONWITH AMNIOTIC FLUID PROTEOMICS SCORE

K Thomas*1, CM Salafia2,3, I Buhimschi4, CS Buhimschi4, E Zambrano4,M Sottile1. 1University of Oregon, United States, 2Placental Analytics LLC,United States, 3Institute for Basic Research, United States, 4Yale UniversitySchool of Medicine, United States

Background: The diagnosis of intraamniotic infection is considered clin-ically relevant to maternal, fetal, neonatal and childhood morbidity andmortality. However, intraobserver variability, even among expert pathol-ogists, remains problematic, apart from 0.96 agreement on ‘‘present vabsent’’ (Pediatr Dev Pathol. 2003;6(5):435-48), with ‘‘consensus’’ (ratherthan valid biomarkers) being the gold standard. An automated andreliable method for quantitation of neutrophils would be a useful diag-nostic tool.Methods: We sampled 10 cases with amniotic fluid proteomics (AFMR)scores, 2 each of scores 0-4 as per Buhimschi et al, BJOG. 2009Jan;116(2):257-67. Two images were taken at random of extraplacentalmembranes; each image was analyzed at both 10x and 20x magnification.The image analytic technique begins with scanned slides in the form ofcolor (RGB) images. We first segment the image to separate tissue fromcells of interest by using the difference in color that results from staining.After this initial segmentation, we then must filter out image features thatare the correct color but fail to fit the expected shape of a neutrophil, suchas [carrie insert name of elongated cells here].This requires the compu-tation of the area, perimeter, and eccentricity (a measure of how circulara shape is) of each connected component identified by the segmenter.Three segmentations were done:1. based on color threshold alone; 2.based on color and then rejecting anything bigger or smaller than the areathreshold interval; 3. based on color, rejecting groups based on areathreshold as well as rejection based on eccentricity (rejecting cells such asfibroblasts that have cigar shaped nuclei).Results: Using Method 1, only the number of pixel groups was associatedwith AF MR (r¼0.494), with Method 2, AF MR was associated with bothpositive pixel count (r¼�0.544), and percent positive pixels (r¼0.544).With Method 3, positive pixel count (r¼�0.546), percent positive pixels(r¼0.546), and the number of pixel groups (r¼0.505) were high correlatedwith AF MR. These counts also significantly correlated with histologicgrading of neutrophils in amnion, chorionic and decidua, and in umbilicalcord. Magnification at analysis did not modify the strength of theassociations.Conclusion: Pilot data suggests that reproducible and reliable automatedsegmentation and quantitation of neutrophils can be performed, withstrong correlations with amniotic fluid proteomic markers of infection andinflammation. We anticipate that a larger image sample per tissue willresult in improved correlation with AFMR score.

Keywords: image analysis, chorioamnionitis, neutrophil, amniotic fluidproteomics

[P10.28].EFFECTS OF GLUCOSE ON THE EXPRESSION OF VEGF SPLICE VARIANTSIN NORMAL AND DIABETIC PLACENTA

F. Sciota*, L. Leach. University of Nottingham, United Kingdom

Hyperglycaemia is a main characteristic of diabetes and may affectplacental vascular development and permeability. In Type1 diabetes, theplacental vasculature displays increased angiogenesis and permeability.The main form of VEGF165 is pro-angiogenic and pro-permeability, whilstVEGF165b, a recently discovered splice variant of VEGF165a, is thought tobe anti-angiogenic, but pro-permeability. Whether glucose can affect theexpression of these variants in the human normal and diabetic placenta isnot known and is the aim of this study.Using chorionic villous explants, 15mM glucose was administered tonormal (N15) and Type1 diabetic (D15) study groups (n¼3 placentae pergroup) for 4h. Euglycaemic controls (n¼3 per group, N5 and D5 respec-tively) contained 5mM glucose. VEGF-165a and VEGF-165b were localisedand counted by immunocytochemistry and selective random sampling.D5 showed downregulation of VEGF165b compared to N5 (p<0.01).Hyperglycaemic insult resulted in a decrease (p<0.01) in vesselsexpressing VEGF165b in N15 compared to N5. This aggravation was notseen in D15. The values of N15 were similar to those in D5 and D15.VEGF165a staining showed increased expression in diabetic explantscompared with normal, with further increases seen with 15mM glucose.VEGF165a upregulation was also seen between N5 and N15 (p<0.001).There was a negative correlation between VEGF165a and VEGF165b(p<0.001;R2¼0.6852).These changes indicate that glucose is able to affect the expression of bothsplice variants of VEGF, with downregulation of the anti-angiogenic splicevariant being a predominant feature. Hyperglycaemia can induce the dia-betic phenotype in normal explants, upregulating VEGF165a and down-regulating VEGF165b. In diabetics, with pre-existing high levels ofVEGF165a and low levels of VEGF165b, hyperglycaemia induced furtherincreases in the pro-angiogenic VEGF165a whilst expression of the splicevariant VEGF165b remain damped. The ratio of the two VEGF splice vari-ants may be an important predictor of vascular dysfunction in diabetes.Funded by ASGBI.Keywords: Diabetes, VEGF-A, VEGF165b


[P10.29].TGFb SIGNALLING VIA PAR6 REGULATES TROPHOBLAST CELL POLARITY

T Sivasubramaniyam*1,2, I Caniggia1,2. 1Samuel Lunenfeld Research Insti-tute of Mount Sinai Hospital, Canada, 2University of Toronto, Canada

Introduction: Cell polarity plays an important role in cell differentiationshaping proper organogenesis. Loss of cell polarity is partly mediatedthrough the TGFb Smad-independent signalling pathway. Activation ofPar6, a key regulator of cell polarity, by TGFb leads to its association withSmurf1 (Smad ubiquitin regulatory factor 1) and to the dissolution of tightjunctions. The contribution of the Smad-independent signalling pathwayvia Par6 to trophoblast cell polarity and differentiation remains elusive.Herein we sought to examine the expression/role of Par6 in normal andpathological placentae.Methods: Expression of Par6 and Smurf1 were examined in placentaethroughout gestation (6-39 weeks) and in preeclamptic (24-31 weeks) andage-matched control tissues (25-34 weeks) using immunoblotting. Par6/Smurf interaction was assessed by co-immunoprecipitation and dual-labeled immunofluorescence. To establish a role for Par6 in regulatingtrophoblast cell polarity Par6 siRNA was employed in choriocarcinomaJEG3 cells and cell polarity markers including Zona occludin-1 (ZO-1) andE-cadherin were tested. Par6 and Smurf1 expression was examined in JEG3cells cultured at 3% and 20% oxygen.Results: Par6 and Smurf1 protein expression and interaction peaked at 10-14 wks of gestation, when oxygen tension increases. Par6/Smurf1 expres-sion also increased in JEG3 cells with increasing oxygenation. Early on ingestation, Par6 localized mainly to the nuclei of cytotrophoblast cells while,with advancing gestation, it shifted to the cytoplasm and it was found at theinterface between cytotrophoblasts and syncytium, where it associatedwith Smurf1, ZO-1 and E-cadherin. Silencing of Par6 resulted in decreasedZO-1 and E-cadherin expression. Of clinical significance, both Par6 andSmurf1 protein expression levels were decreased in preeclampsia.Discussion: Par6 plays a role in regulating trophoblast cell polarity duringplacental development. These findings provide novel insights into the roleof Smad-independent signalling with respect to cell polarity in the path-ogenesis of preeclampsia. (Supported by CIHR and OWH/IGH).Keywords: cell polarity, preeclampsia, oxygen regulation, development

[P10.30].EVIDENCE OF ABNORMAL PLACENTAL LYMPHATIC DEVELOPMENT INA CASE OF PLACENTAL MESENCHYMAL DYSPLASIA

SD Smith*1, N Sahasrabudhe2, EA Martindale3, AEP Heazell1. 1Maternal andFetal Health Research Group, United Kingdom, 2Department of Histopa-thology, Royal Blackburn Hospital, Blackburn, United Kingdom, 3Depart-ment of Obstetrics and Gynaecology, Royal Blackburn Hospital, Blackburn,United Kingdom

Placental mesenchymal dysplasia (PMD) is a rare disorder affecting 0.02%of human pregnancies. PMD is associated with stillbirth, intrauterinegrowth restriction (IUGR) and Beckwith-Wiedemann syndrome (animprinting disorder characterised by elevated insulin-like growth factor(IGF)-II expression). 86 cases of PMD have been described and itsmorphology is characterised by placentomegaly and the presence ofvesicles, similar to those seen in molar pregnancies. The underlyingcellular origin of this condition is unclear. We investigated the placentalcell type involved in a case of PMD associated with a live born femaleinfant with IUGR. In this case, intermediate and terminal villi containedcisternae, lined by non-proliferative cells, as verified by immunohisto-chemistry for Ki67. Immunostaining for cytokeratin-7 and CD-34 revealedthat these cisternae were not of trophoblast nor vascular endothelialorigin. However, the cellular lining of the cisternae exhibited positiveimmunostaining using antibodies specific for lymphatic endothelialmarkers: CCL21, vascular endothelial growth factor (VEGF) receptor 3 andD2-40. No staining was detected in the intermediate or terminal villi ofnormal third trimester pregnancies. This aberrant lymphangiogenesis is inaccordance with current hypotheses regarding the potential role of VEGF-D and IGF-II in the aetiology of PMD, both of which can induce lym-phangiogenesis in vitro. Furthermore, such observations suggest thatplacental villous mesenchyme has the potential to differentiate intovarious cell types, including those not normally present in the term humanplacenta.Keywords: Placenta, IUGR, Mesenchyme, Lymphangiogenesis


[P10.31].CATCH-UP GROWTH OF PLACENTAS IN LATE-ONSET PREECLAMPTICPREGNANCIES?

D.U. Ulrich*1, G.D. Desoye1, C.W. Wadsack1, U.W. Lang1, J.H. Haas1,D.S. Schlembach1,2. 1Department of Obstetrics and Gynecology, MedicalUniversity of Graz, Austria, 2Prenatal Center Muenchen, Germany

Background: Placental weight has been correlated with fetal outcome,morbidity and mortality and even with diseases in later life. Although thedynamics of placental growth as reflected by weight changes has beendocumented in few studies and placental weight percentile curves wereestablished for uncomplicated singleton pregnancies, similar data forpregnancy pathologies have not been available. The aim of our study wasto establish placental weight percentiles for pregnancies complicated bypreeclampsia (PE) to calculate weight increases and to compare theseresults with normal pregnancies.Methods: In this retrospective analysis the data of 4834 singleton preg-nancies were analysed with deliveries �28 week of gestation (wks).Among these 197 (4%) were PE pregnancies (defined by RR >140/90,proteinuria >0,3 g/ 24 hrs). All placentas were weighted within 10 minutesafter their delivery and after cutting the umbilical cord. Growth dynamicswas calculated as difference in placental weights along 4-week intervals.Results: In both groups the mean placental weight showed a steadyincrease throughout pregnancy. As expected the preeclamptic placentaweight was significantly lower compared to the control group. Bothabsolute and relative weight difference between normal and preeclampticplacental weight decreased from early to late pregnancy: at 32 wks -134 g(-31%), 36 wks -90 g (-17%) and at 40 wks -62 g (-11%). Placental weightdifference between wks 28-40 and 32-40 were higher in PE (+65%, +46%)than in normal pregnancies (+35%, +28%), whereas fetal weight differenceswere virtually similar in both groups.Conclusion: Although placental weight is lower in PE than in normalpregnancies in the third trimester of pregnancy, placentas in PE gain moreweight in this period than in normal pregnancies. This is not accounted forby a similar catch-up growth of the fetuses.Keywords: placenta weight percentile, preeclampsia, placenta weight gain

[P10.33].IMPAIRED SYNCYTIALISATION IN SEVERE PRETERM IUGR

K Widdows*1, S Drewlo2, J Kingdom2, T Ansari1. 1Dept. of Surgical Research,NPIMR, Harrow, London, United Kingdom, 2Dept. Obstetric & Gynaecology,SLRI, Mount Sinai Hospital, Canada

The small placental phenotype of severe preterm IUGR is characterized bysignificant reductions in fetoplacental blood flow, resulting from increasedvascular impedance from poorly developed peripheral villous capillariessecondary to a defective angiogenic drive in the late second trimester ofpregnancy. Molecular evidence suggests this diminished growth potentialarises at the level of villous trophoblast differentiation. We previouslyreported using stereological tools that the poorly developed villi of severeIUGR with abnormal umbilical artery Doppler contain 30% fewer prolif-erating villous cytotrophoblast cells (vCT), reflecting premature loss ofvillous progenitor cells that can differentiate, fuse and regenerate theoverlying syncytium throughout gestation. This depletion however did notreduce the relative numbers of vCT, indicating an imbalance in the relativeproportions of proliferating vs. differentiating vCT- favouring a ‘differenti-ating’ vCT phenotype. We therefore tested the hypothesis that in severeIUGR, impaired vCT differentiation leads to the focal depletion of syncy-tiotrophoblast nuclei, using stereological analysis. The morphological basisof syncytial fusion was investigated by estimating (i) the relative and totalnumber of syncytiotrophoblast nuclei and (ii) the mean individual (size)and overall volume of syncytial knots as an index of syncytial extrusionand apoptosis, in five preterm normotensive IUGR and nineteen IUGR withpre-eclampsia (PET) cases with documented abnormal umbilical arteryDoppler. Whilst total numbers of SCT were significantly reduced incomparison to preterm controls (n¼12), the relative numbers were furtherreduced by 50% in both IUGR and IUGR-PET. This focal depletion insyncytial nuclei alongside ‘normal’ numbers of vCT (non-fusing) impliesfocal impairment of syncytial fusion. Furthermore, syncytial knot volume,size and apoptosis were increased. Our data indicates that impairedsyncytial fusion indicative of defective vCT differentiation results ininsufficient numbers of syncytiotrophoblast nuclei needed to maintainadequate nutrient transport to the severely growth restricted fetus inde-pendent of the small placental phenotype.


[P10.34].A CASE OF TWIN PREGNANCY WITH COMPLETE HYDATIDIFORM MOLEAND COEXISTENT FETUS: MORPHOLOGICALLY NORMAL PLACENTAWITH TRIPLE TRISOMY

M. Yamaguchi*, K. Uchino, J. Miyoshi, H. Tashiro, T. Ohba, H. Katabuchi.Kumamoto University, Japan

A 27-year-old nulliparous woman at 11 weeks of gestation was referred toour University Hospital because of unusual ultrasound findings of theconceptus. Ultrasonography showed multiple cysts within the placenta inthe presence of the living fetus. The patient’s serum hCG level was 462,200mIU/ml. On the basis of these findings, partial hydatidiform mole, andplacenta with hydropic change were considered in the differential diag-nosis. The hCG level was continually elevated and peaked at 900,000 mIU/ml. Each placental cystic lesion enlarged in size during the follow-upperiod. We performed MRI scan at 15 weeks of gestation. The MRI findingsshowed a clear distinction between the normal placenta and the multi-vesicular tissue. A diagnosis of hydatidiform mole coexisting with a fetuswas made. We informed the patient about the high risk of maternalcomplications and sequelae. Consequently, she decided to terminatepregnancy at 15 weeks of gestation. The evacuated vesicular tissue andmorphologically normal placenta were histophathologically examined.The diagnosis of hydatidiform mole was confirmed and the placenta wasconfirmed as normal. The karyotype of the molar tissue was 46,XX and thatof the histophathologically normal placenta was 49,XX,+2,+12,+16. Cyto-genetic examination showed that the genome of the complete hydatidi-form mole were androgenetic homozygous diploid. It is postulated thata dispermic triploid zygote may divide into a normal biparental cell anda cell with a haploid set of paternal chromosomes. The latter cell coulddevelop into a complete mole by diploidization. A tripolar spindle may beformed at the time of the first cleavage division, resulting in an aberrantchromosomal distribution, which may affect the diploid fetus. This casecan support the hypothesis of postzygotic diploidization of triploids.Keywords: hydatidiform mole and coexistent fetus, postzygotic diploid-ization of triploids, chromosomal aberration, molar pregnancy

[P11.01].CONDITIONED MEDIUM OF PLACENTAL MULTIPOTENT MESENCHYMALSTROMAL CELLS PROTECTS ENDOTHELIUM FROM OXIDATIVE INJURYVIA STAT3 AND MANGANESE SUPEROXIDE DISMUTASE ACTIVATION

C.-P. Chen*1, J.-P. Huang1, Y.-Y. Chen1, Y.-H. Wu2, C.-Y. Chen2, S.-H. Liu2.1Division of High Risk Pregnancy, Mackay Memorial Hospital, Taiwan,2Department of Medical Research, Mackay Memorial Hospital, Taiwan

We hypothesized that endothelial cells undergone oxidative injury inducedby reactive oxygen species could be repaired by the paracrine factors ofhuman placental multipotent mesenchymal stromal cells (hPMSCs). Thealterations of antioxidant enzyme activities and mechanisms of anti-apoptotic effects on endothelial cell induced by the conditioned medium ofhPMSCs were studied. hPMSCs were isolated from term placentas andendothelial cells from umbilical veins. A conditioned medium of hPMSCswas harvested. Medium conditioned by hPMSCs supported endothelial cellsurvival and enhanced endothelial cell against tert-Butyl hydroperoxideinduced intracellular peroxides and apoptosis. RT-PCR revealed hPMSCexpressed cytokines of IL-6 family and the receptors of these cytokines inendothelial cells. The conditioned medium activated the expression andtranscriptional activity of signal transducer and activator of transcription 3(STAT3) in endothelial cells. The endothelial cell STAT3 expression andtranscriptional activity was inhibited by gp130 neutralizing antibody addedin the conditioned medium. The anti-apoptotic effect of conditionedmedium was further inhibited when the endothelial cells was transfectedby STAT3 small interfering RNA. Manganese superoxide dismutase, but notcopper/zinc superoxide dismutase, catalase or glutathione peroxidase ofendothelial cell was significantly up-regulated by conditioned mediumboth at mRNA transcript and protein levels, which was mediated by theactivation of STAT3 and was inhibited by the gp130 neutralizing antibody orSTAT3 small interfering RNA. Thus, the paracrine factors secreted byhPMSCs may support endothelial cell survival. The activation of STAT3 andmanganese superoxide dismutase induces a protective effect on oxidativestress-induced endothelial cell damage.Keywords: conditioned medium, multipotent mesenchymal stromal cells,manganese superoxide dismutase, STAT3


[P11.02].MESENCHYMAL STEM CELLS DERIVED FROM HUMAN PLACENTA–ISOLATION, IN VITRO EXPANSION AND CHARACTERIZATION

L. Danisovic*1, M. Ulicna1, I. Varga1,2, V. Repiska1, D. Bohmer1, S. Polak1.1Comenius University, Faculty of Medicine, Slovakia, 2Slovak MedicalUniversity, Slovakia

Background: Stem cells are generally characterized as clonogenic,undifferentiated cells with unique self-renewal potency and plasticity.Over the past few years, stem cells have been derived from various tissuesof embryonic, fetal and adult origin. Embryonic stem cells are consideredpluripotent, but their utilization is restricted by the ethical consideration.For that reason, multipotent adult stem cells also referred as mesen-chymal stem cells (MSCs) represent exclusive tool for regenerativemedicine. Human placenta also belongs to promising source of MSCs. Themain goal of present work was isolation, in vitro expansion andmorphological as well as biological characterization of human placenta-derived MSCs.Material and Methods: Human placentas (n¼5; normal pregnancies)were obtained from healthy donors, always following patient’s informedconsent. Samples of tissue (1cm3) from placenta lobules were cut intosmall fragments, vigorously rinsed in phosphate buffered saline, hemo-lyzed and digested with 0.25% trypsin for 15 min at 37�C. Obtained cellsuspension with residual fragments was filtered through 70 mm cellstrainer. After centrifugation (1200 rpm for 10 min), collected cells weresuspended in a-minimum essential medium with 10% fetal bovine serumand gentamicin (80 mg/ml). Cultures were expanded up to third passage at37�C in humidified atmosphere with 5% CO2. Cell culture medium wasrefreshed twice a week. Cells from third passage were analyzed by lightand transmission electron microscope (TEM), the analyses of surfacemarkers were performed by FACS. Moreover, the multilineage potentialwas examined as well.Results and Conclusion: Light microscopy showed that MSCs derived fromplacenta had fibroblast-like morphology. Subsequent TEM observationshowed typical ultrastructure of MSCs. Almost all of the analyzed cells wereCD29, CD44, CD90, CD105, CD166 positive and CD34, CD45 negative. They didnot express anti–human fibroblast surface protein. Moreover, the chondro-genic and osteogenic differentiation was proved. In conclusion, humanplacenta represent high throughput source for mesenchymal stem cells.Keywords: placenta, mesenchymal stem cell, in vitro expansion,characterization

[P11.03].IN VITRO CHARACTERIZATION OF YOLK SAC MEMBRANE FROM EQUINE

ALR Franciolli*1, BM Cordeiro2, AC Morini1, JC Morini-Junior1, CV Wence-slau1, MA Miglino1. 1Department of Surgery, Faculty of Veterinary Medi-cine, University of Sao Paulo, Sao Paulo, Brazil, 2Presbiteriana MackenzieUniversity, Sao Paulo, Brazil, 3Department of Morphology, UNIfeob, SaoJoao da Boa Vista, Sao Paulo, Brazil

The yolk sac is the first of the fetal membranes that develops in eutherianmammals. It is attached to the wall of exocelom and extra-embryonicmesoderm. The aim of this study was to characterize the morphology ofthe equine yolk sac cells in culture with 20, 30 and 40 days of gestation inorder to demonstrate its multipotentiality and its ability to differentiate indifferent lineages. The yolk sac explants were cultivated in DMEM-H with20% FBS Hyclone and 1% penicillin/streptomycin. The culture flasks weremaintained at 37�C in a humidified environment containg 5% carbondioxide. After expansion, the cells were morphologically analyzed byinverted microscopy (NIKON ECLIPSE, TS100). The cultures of equine yolksac cells with 20, 30 and 40 days of gestation were composed of numerousundifferentiated cells floating, with circular format at the first 24 hours ofculture. Followed the first five days of culture, the medium of culture wasdiscarded along with the floating cells, leaving only cells that have capacityfor adhesion to substrate. Among the different cell types in culture byfragments released yolk in both ages, we found cells with fibroblast-likecharacteristics and with characteristic epithelial and less frequently ovalcells. Morphologically the cells with fibroblast-like appearance were smallformat with elongated, fusiform and reduced cytoplasm. The epithelial-looking cells were large and rounded, with regular cytoplasmic membrane,cytoplasm, and large sparse, small, circular core. As preliminary conclu-sions, we believe that in the three gestational ages (20, 30 and 40) the yolksac exhibits two distinct types of cell populations, but new knowledgerelated to the capacity of differentiation are under experiment to confirmthe pluripotential cells of the yolk sac.Keywords: Yolk sac, Equine, Stem cells, Culture


[P11.05].AMNION EPITHELIAL CELL ISOLATION FOR USE IN CLINIC

S Murphy*, S Rosli, R Acharya, R Lim, G Jenkin, E Wallace. MonashUniversity, Australia

Introduction: Human amnion epithelial cells (hAECs) are a heterogeneouspopulation positive for stem cell markers and display multi-lineagedifferentiation potential, differentiating into cells of the endoderm (liver,lung epithelium), mesoderm (bone, fat), and ectoderm (neural cells). Theyhave a low immunogenic profile and possess potent immunosuppressiveproperties. Hence hAECs may be a valuable source of cells for cell therapy.Aim: Demonstrate feasibility of animal product-free methods of isolation andculture according to current guidelines on cell preparation for clinical use.Methods: An animal product-free cell isolation method was developedand compared to traditional animal product-containing methods. Total cellyield and viability determined by cell counts and trypan blue exclusion.Purity was established through FACS analysis of epithelial (EpCAM) andmesenchymal (CD90, CD105) marker expression. Animal product-freecryopreservation and growth media were developed and compared toconventional serum-based media. Post-thaw viability, recovery of cellmetabolism, and proliferation rates were determined. hAECs were ana-lysed after 5 passages by karyotype analysis, cell cycle distribution andchanges to telomere length. hAEC were differentiated into lineages of thethree primary germ layers and specific markers analysed using PCR,immunocytochemistry and histology.Results: The method developed was comparable to established animalproduct-containing methods, producing an average yield of 120�40x106

hAECS with average viability of 83�4%. Isolated populations were 92%EpCAM positive with <1% mesenchymal cell contamination. After 5passages hAEC displayed normal karyotype, cell cycle distribution and longtelomeres, suggesting that hAEC are unlikely to be tumorigenic. Multi-potent differentiation potential of hAEC was demonstrated by induction ofneural (MAP2, GFAP, Nestin), bone (osteocalcin, osteonectin), fat (PPARgLPL), and lung epithelial (SP-C, CC10, Nkx2.1) gene expression as well as byimmunocytochemical and histological methods.Discussion: We have now optimised an animal product-free method forefficient isolation and cryopreservation of hAECs suitable for clinicaltherapies.Keywords: amnion, epithelial, isolation, differentiation

[P11.06].Ly6e EXPRESSION IS RESTRICTED TO SYNCYTIOTROPHOBLAST CELLS OFTHE MOUSE PLACENTA

M Hughes, DRC Natale*. University of Calgary, Canada

The labyrinth layer of the mouse placenta is primarily derived from cells ofthe chorion and is the site of nutrient and gas exchange between maternaland fetal blood. Organized in a fixed orientation, three layers of tropho-blast followed by a layer of fetal endothelia separate the maternal and fetalblood spaces. A mononuclear trophoblast giant cell layer lines thematernal blood spaces, followed by two multi-nucleated syncytiotropho-blast layers (SynT I and II respectively). It has been shown previously, thatSynT I and II cells can be identified by the layer-restricted expression ofSyncytin a and Gcm1, respectively. In addition, these genes are expressed ina similar, spatially related manner in the chorion, suggesting pre-patterning of labyrinth development. In the present study, we character-ized the expression of lymphocyte antigen 6, locus E (Ly6e), in the mouseplacenta. Ly6E is a membrane-associated protein that is expressed in thehematopoietic lineage and is a marker of T-cell precursors. We identifiedLy6e mRNA expression in trophoblast stem (TS) cells in a gene expressionscreen. Northern blot analysis confirmed that Ly6e was expressed in bothundifferentiated and differentiated TS cell cultures as well as placentalRNA from embryonic day (E) 10.5 to 18.5. FACS analysis indicated that invitro, Ly6e was expressed in 33% of undifferentiated TS cells and increasedfollowing differentiation. Interestingly, in vivo, Ly6e was first detectable bymRNA in situ hybridization in a subset of cells in the chorion beginning atE8.5. This pattern of expression differed from Gcm1 but was similar to thatof Syncytin a, and at later stages of gestation, Ly6e expression was restrictedto syncytiotrophoblast cells in the labyrinth. To date, Ly6e representsa novel marker of syncytiotrophoblast cells. Ongoing experiments,utilizing siRNA knockdown of Ly6e in TS cells will determine if it has a rolein syncytiotrophoblast differentiation.Keywords: syncytiotrophoblast, trophoblast stem cell, labyrinth, mouse


[P11.07].INACTIVATION OF INTEGRIN ALPHA 1 (ITGA1) IN MESENCHYMAL STEMCELLS DERIVED FROM THE HUMAN PLACENTA

RA Pace*1,2, P Murthi1,2, N Castrechini1,2, SP Brennecke1,2, B Kalionis1.1University of Melbourne, Department Obstetrics & Gynaecology,Australia, 2Department of Perinatal Medicine, Pregnancy Research Centre,Royal Women’s Hospital, Parkville 3052, Australia

Introduction: Cultured mesenchymal stem cells derived from humanplacenta (PMSCs) are intensely studied because of their potential utility inregenerative medicine [1]. Our interest is in the potential contribution ofPMSCs to the significant pregnancy disorder of fetal growth restriction(FGR). In preliminary work (Castrechini et al, unpublished data), the geneITGA1 showed altered expression in PMSCs from FGR-affected placentaecompared with control term PMSCs. ITGA1 is an important cell adhesionmolecule. In MSCs, ITGA1 regulates proliferation and is used as a colonyforming unit (CFU) marker [2,3]. Here, we used siRNA technology to reduceexpression of ITGA1 in PMSCs.Methods: Term placentae were obtained from healthy mothers withinformed consent. PMSCs were obtained by mechanical and enzymaticdigestion of chorionic villi, and plating onto plastic in specialised cellculture medium [4]. FACS analysis was performed on PMSCs with posi-tive (CD105, CD73) and negative (CD45) markers [5]. Validated shortinterfering RNAs (siRNA) were used to reduce ITGA1 expression inPMSCs. Relative ITGA1 mRNA levels were determined by real-time PCRanalysis.Results: FACS revealed 95-98% of PMSCs were positive for CD73 andCD105, and negative (<1%) for CD45. PMSCs were transfected with twosiRNAs (ITGA1-si1 and ITAG1-si2). Real-time PCR analysis showed a signif-icant relative reduction of ITGA1mRNA with both siRNAs [99.55%�0.0018SEM (si1) and 99.34%�0.0013 SEM (si2)]. Apoptosis markers Bcl2 and Baxwere tested by real-time PCR and there were no significant differencesbetween the siRNAs (ITGA1-si1 and ITAG1-si2) and the negative controlsuggesting that reduced ITGA1 expression did not alter expression of Bcl2and Bax.Discussion: This is the first study to successfully knockdown geneexpression in PMSCs. Current studies are investigating the role of ITGA1 incell migration, proliferation and adhesion.Bibliography:1. Wulf, G.G., et al., Tissue Eng, 2004. 10(7-8): p. 1136-47.2. Rider, D.A., et al., J Mol Histol, 2007. 38(5): p. 449-58.3. Stewart, K., et al., Cell Tissue Res, 2003. 313(3): p. 281-90.4. Battula, V.L., et al., Differentiation, 2008. 76(4): p. 326-36.5. Brooke, G., et al., Br J Haematol, 2009. 144(4): p. 571-9.Keywords: Placental stem cells, FGR, Integrin

[P11.08].PPARg REGULATES DIFFERENTIATION AND sFLT EXPRESSIONDOWNSTREAM OF HYPOXIA IN MOUSE TROPHOBLAST STEM CELLS

V Tache*1, A Ciric1, DS Milstone2, MM Parast1. 1University of California SanDiego, United States, 2Brigham and Women’s Hospital, United States

PPARg is a ligand-activated transcription factor involved in many cellularprocesses, including inflammation, metabolism, and differentiation.PPARg-null embryos die at midgestation due to placental abnormalities,the most severe of which is lack of formation of the labyrinth. We havepreviously derived trophoblast stem (TS) cells from both wild-type (WT)and PPARg-null mouse embryos. PPARg-null TS cells differentiateprematurely, and exclusively, into trophoblast giant cells (TGC), showingthat PPARg is necessary for differentiation into labyrinthine trophoblast. Inthe current study, we investigated the relationship between oxygentension and PPARg expression, since hypoxia inhibits trophoblast differ-entiation and is a causative factor in many obstetric complicationsinvolving placental dysfunction. Using qPCR analysis of a panel of markers,we evaluated differentiation patterns of WT and PPARg-null TS cells underboth normoxia and hypoxia (2% oxygen). Our results show that PPARgexpression is turned on when TS cells are switched to differentiationmedia, and is downregulated under hypoxia. In addition, PPARg was notrequired for hypoxia-induced inhibition of giant cell differentiation;however, when reintroduced into WT-TS cells differentiating underhypoxia, PPARg induced differentiation specifically into labyrinthinetrophoblast, as shown by upregulation of Gcm1 and syncytin-A. PPARg-agonist (rosiglitazone) treatment of WT-TS cells inhibited giant celldifferentiation under normoxia, and also specifically inhibited hypoxia-reoxygenation-induced expansion of spongiotrophoblast. Finally, PPARg-null TS cells showed highly upregulated sFlt expression, and rosiglitazonetreatment of TS cells decreased sFlt secretion in a PPARg-dependentmanner. We propose a model where HIF-induced downregulation ofPPARg leads to inhibition of labyrinthine trophoblast differentiation andenhanced expression of sFlt, which has been associated with developmentof pre-eclampsia. We suggest PPARg as an excellent therapeutic target forhypoxia-associated obstetric complications, including pre-eclampsia andfetal growth restriction.Keywords: PPAR-gamma, Hypoxia, sFlt, trophoblast stem cells


[P11.09].HOMEOBOX GENE EXPRESSION IN PLACENTAL MESENCHYMAL STEMCELLS

S Qin*1,2, RA Pace1,2, P Murthi1,2, SP Brennecke1,2, B Kalionis2. 1University ofMelbourne, Department Obstetrics & Gynaecology, Australia, 2Departmentof Perinatal Medicine, Pregnancy Research Centre, Royal Women’sHospital, Parkville 3052, Australia

Introduction: Mesenchymal stem cells (MSCs) are widely studied for theirpotential in regenerative medicine due to their ability to differentiate intomultiple cell types and avoid allograft rejection. In embryonic stem cells,many important regulatory genes including the homeobox genes NANOGand OCT4 have been identified. Few studies of regulatory genes in MSCshave been reported. Previously in our laboratory, we screened forhomeobox genes in placental MSCs (PMSCs) and identified 5 novelhomeobox genes, DLX5, TGIF, MEIS2, HLX, and HEX, by real-time PCR. Theobjective is to examine the protein expression of these genes in PMSCsusing immunocytochemistry.Method: The MSCs for this study were isolated by enzymatic digestionfrom normal term placentae (37-41 weeks, n¼5). Expression of stem cellmarkers on cultured PMSCs was verified by flow cytometry using the MSCpositive markers CD105, CD73 and negative marker CD45. Immunocyto-chemistry was carried out with the appropriate primary antibody andcolour detection was by AEC red.Results: PMSC preparations showed characteristic fibroblast-likemorphology and were capable of forming CFU-F colonies. Greater than 90%of the cells expressed the markers CD105 and CD73, and less than 2% of thecells express CD45. DLX5 and TGIF show expression exclusively in thenucleus whereas in MEIS2, HLX, and HEX were predominantly expressed inthe nucleus but also showed cytoplasmic expression.Discussion: We have confirmed at the protein level that novel PMSChomeobox genes are expressed in the nuclei of PMSCs. The role of thehomeobox genes in PMSCs is not known, however TGIF and MEIS2 playroles in MSCs in other tissues. HLX is of particular importance since wehave studied this gene extensively in the placental trophoblast cells whereit regulates cell proliferation and migration but its role in PMSCs has notbeen determined. We are optimizing conditions for HLX inactivation inPMSCs and will investigate the effect on PMSC functions.Keywords: Placental stem cells, homeobox genes, FGR

[P11.10].FETAL-DERIVED ENDOTHELIAL PROGENITOR CELLS ARE SEQUESTEREDBY THE HUMAN PLACENTA

P Sipos*, M Wareing, I Crocker, P Baker. Maternal and Fetal RedearchCenter, The University of Manchester, United Kingdom

Introduction: Endothelial Progenitor Cells (EPCs) are circulating cellsproduced by the bone marrow in the adult, which contribute to vascularrepair and neovascularisation. There are two subtypes: Endothelial ColonyForming Cell (ECFCs), which are highly proliferative and develop into matureendothelial cells, and Circulating Endothelial Cells (CACs), haemopoietic cellswhich migrate into the intima of the forming vessel and regulate ECFCfunction. It is unknown whether EPCs play a role in placental vasculogenesis.It is also unknown whether EPC are produced by the placenta or the fetus.We therefore aimed to investigate whether EPCs migrate to the placentafrom the fetus and become incorporated into placental vessels.Methods: We counted CAC and ECFC numbers in both arterial and venousumbilical blood from 10 newborns from uncomplicated pregnancies by 5 colourflow cytometry(1). ECFCs were recorded as CD31bright/CD45-/KDR+/CD34+ andCACs as CD31+/CD45/CD133+/CD34+. FcR blocker was used to prevent non-specific binding and 7AAD to exclude non-viable cells. ECFCs and CACs werefurther expanded from fetal blood(2), trackered and perfused ex vivo intoplacental chorionic arteries, before culturing for 48 hours. Snap frozen, OCT-embedded vessel sections were subsequentlycut to determine EPC-localisation.Results: Numbers of CACs and ECFCs (as percent of mononuclears) weresignificantly higher in the umbilical artery than vein (CAC:0.375�0.388 vs.0.293�0.306, median�SD, p<0.01 Wilcoxon-paired t-test, n¼8;

ECFC:3.089x10-3�8.963x10-3 vs. 2.760x10-4�4.438x10-3, median�SD,p<0.05, n¼10). In perfused chorionic vessels, trackered ECFCs were shownto incorporate into the endothelial layer, whilst CACs were shown to invadethe vessel wall and embed behind the endothelial lining and vascular intima.Discussion: These results suggest that fetal-derived EPCs (both ECFCs andCACs) migrate into the human placenta, where they play a potential role inplacental vascularisation.(1) Duda et al. Nat Protoc. 2007;2:805.(2) Lin Y et al. J Clin Invest. 2000;105:71.This study was supported by the Wellcome Trust.Keywords: Endothelial progenitor cell, sequestration, placenta,vasculogenesis

[P12.02].THE EFFECTS OF PLACENTAL MALARIA PARASITE AND MONOCYTEPRODUCTS ON SYSTEM A-MEDIATED AMINO ACID TRANSPORT INBeWo CELLS

EH Aitken*1, P Boeuf1, J Glazier2, S Rogerson1. 1Department of Medicine(RMH), University of Melbourne, Parkville, Australia, 2Maternal FetalHealth Research Group, University of Manchester, St Mary’s Hospital,Manchester, United Kingdom

The pathogenetic mechanisms underlying the fetal growth restrictionassociated with placental malaria are unknown. However, in a pilot ex-vivo study of malaria-infected and control placentas from Malawi, weshowed that low transcript levels of SNAT-1 (an isoform of amino acidsystem A transporter) were associated with placental-malaria, inter-villositis and low birth weight (LBW).We therefore hypothesized that placental malaria infection and subsequentintervillositis leads to a decreased activity of system A amino acid transportersin the syncytiotrophoblast resulting in fetal growth restriction and LBW.To model the effect of placental malaria and intervillositis on system Aactivity, BeWo choriocarcinoma cells (as a model of placental trophoblast)were exposed to supernatant from a monocyte/malaria parasite co-cultureor to cytokines previously reported to be associated with placental malaria.System A activity was measured as the uptake of 14C-labelled methyl-aminoisobutyric acid (MeAIB). As IL-1b has been associated both withdecreased system A activity and LBW in placental malaria, we measured IL-1b levels in placental serum from malaria-infected and uninfected womenby ELISA, and related these to birth weight and pregnancy outcome.BeWo cells exposed to supernatant from a monocyte/parasite co-culturehad decreased system A activity compared to the control. Exogenous IL-1bwas effective in decreasing system A activity in BeWo cells, whereas othercytokines associated with placental malaria and LBW, including IL-8 andTNF-a, were not. IL-1b was increased in placental serum of women withplacental malaria and intervillositis and IL-1b concentration was nega-tively associated with birth weight in this group.Taken together, these findings suggest a pathogenetic mechanism for fetalgrowth restriction in placental malaria whereby malaria parasites andmonocytes sequestered in the placenta lead to reduced transplacentalamino acid transport, partly due to IL-1b secretion by monocytes, therebyreducing amino acid provision to the fetus.Keywords: malaria, low birth weight, intervillositis, amino acid trans-porter


[P12.03].EXPRESSION, LOCALISATION AND REGULATION OF ATP-BINDINGCASSETTE TRANSPORTERS A1 AND G1 IN HUMAN GESTATIONALTISSUES AND DIFFERENTIATING TROPHOBLASTS

IL Aye*, BJ Waddell, PJ Mark, JA Keelan. University of Western Australia,Australia

Objectives: The ATP-Binding Cassette (ABC) transporters ABCA1 andABCG1 maintain cellular lipid homeostasis by transporting cholesterol,phospholipids and sphingolipids, as well as limiting the entry of toxicoxysterols. As major lipid regulators, these transporters have been impli-cated in diverse cellular processes including cell differentiation andapoptosis. Loss of ABCA1 function in mice leads to placental malformation,perturbed steroidogenesis, intrauterine growth restriction and neonataldeath. Recently, efflux of cholesterol via ABCA1 and ABCG1 was demon-strated in fetal endothelial cells of the human placenta. In this study, theexpression of ABCA1 and ABCG1 was investigated in human term placentaland extra-embryonic tissues. Furthermore, the expression and regulationof these transporters was also investigated in differentiating primarytrophoblasts in vitro to determine their role in placental development.Methods & Results: Using quantitative PCR and immunoblotting analyses,ABCA1 and ABCG1 expression was detected in placenta and fetalmembranes (amnion and choriodecidua). ABCA1 and, to a lesser extent,ABCG1 were localised by immunofluorescence to the apical syncytio-trophoblast membrane. Endothelial cells of terminal villi also stained forABCA1, while ABCG1 was prominent in vessels of stem villi. Both trans-porters were also present in amniotic epithelium, chorion and decidualtissues. ABCA1 and ABCG1 mRNA and protein expression increased withdifferentiation after 5 days in culture (approximately 2-6 fold), consistentwith either an involvement in the syncytialisation process or a phenotypicexpression marker. In culture, expression of both transporters in placentaltrophoblasts was stimulated by THE liver X receptor ligand T0901317whereas peroxisome proliferator-activated receptor (PPARa and PPARg)ligands (GW7647 and rosiglitazone) and proinflammatory cytokines (IL-1band TNF-a) were ineffective.Conclusion: These findings suggest that ABCA1 and G1 mediate cholesteroldelivery to both the fetal and maternal circulation, in addition to as-yetundefined roles in fetal membranes.Keywords: Transporter, Cholesterol, Differentiation, Lipid

[P12.04].DIFFERENTIAL IMPACT OF MATERNAL FOOD RESTRICTION ON AQP1AND AQP8 EXPRESSION IN RAT PREGNANCIES AT E16

L Belkacemi*, MH Beall, JT Lin, Q Liu, M Desai, MG Ross. Dept. of Obstetricsand Gynecology, David Geffen School of Medicine at UCLA and LABIOMEDResearch Institute at Harbor-UCLA Medical Center, CA, United States

Introduction: Transplacental water flow, essential for maintenance of fetalbody water and amniotic fluid (AF), may be regulated in part by aquaporin(AQP) channels. Maternal food restriction (MFR) during pregnancy results inintrauterine growth restriction (IUGR) and IUGR is associated with oligo-hydramnios. Given the potentially crucial role of water flow in the placenta,we determined the effect of MFR during pregnancy on AF volume (AFV) andthe placental expression of AQP1 and AQP8. We focused our study on thetwo placental positions (proximal and mid-horns) with the extremes ofnutrient/oxygen supply, and the two distinct placental zones (basal, site ofhormone production; and labyrinth, site of feto-maternal exchange).Methods: Pregnant rats were fed an ad libitum diet (AdLib) or were 50%MFR beginning at E10. At E16 rats were euthanized and gestational sacsfrom left and right mid- and proximal horns dissected. AFV was quantifiedas the difference in sac weight before and after drainage. AF osmolality wasdetermined by freezing point depression. The placenta and fetus wereseparated and weighed. Six placentas from right mid- and proximal hornswere separated into basal and labyrinth zones and analyzed for AQP1 andAQP8 protein expression.Results: MFR fetal weights from the proximal and mid-horns were notsignificantly different from their respective AdLib. MFR placental basalzone weights from mid-horn placentas were significantly decreasedcompared to AdLib. In contrast, MFR placental labyrinth zone weights fromboth mid- and proximal horns were not significantly different from theirrespective AdLib. AFV was significantly lower in the MFR from mid- andproximal horn sacs. Inversely, AF osmolality was significantly increased inMFR from mid- and proximal horn sacs. AQP1 protein decreased signifi-cantly in MFR basal zone from proximal horn and MFR labyrinth zone frommid- and proximal horn placentas. Inversely, AQP8 was increased in MFRbasal and labyrinth zones from mid- and proximal horn placentas.Conclusion: MFR results in decreased AFV and increased osmolality priorto significant fetal growth restriction. This change was accompanied witha reduction in AQP1 protein in the placenta suggesting a reduction in waterflow. The increase seen in AQP8 expression may result in increasedplacental flow of solutes rather than water since AQP8 transport bothwater and ammonia.Keywords: Aquaporin, pligohydramnios, placenta, water


[P12.05].ZONAL AND POSITIONAL IMPACT OF MATERNAL FOOD RESTRICTIONON PLACENTAL AQP1 AND AQP8 EXPRESSION AT TERM GESTATION INRAT

L Belkacemi*, MH Beall, JT Lin, Q Liu, M Desai, MG Ross. Dept. of Obstetricsand Gynecology, David Geffen School of Medicine at UCLA and LABIOMEDResearch Institute at Harbor-UCLA Medical Center, CA, USA, United States

Introduction: Transplacental water flow, essential for the maintenance offetal body water and amniotic fluid (AF), may be regulated in part by certainaquaporin (AQP) channels. Maternal food restriction (MFR) during pregnancyresults in intrauterine growth restriction (IUGR) and IUGR is associated witholigohydramnios. Therefore, we hypothesize that impaired placental waterflow in MFR results from dysregulation of placental AQP proteins. Weassessed the effect of MFR during pregnancy on AF volume (AFV) andplacental expression of AQP1 and AQP8 at E20. We focused our study on twouterine positions (proximal and mid-horns) with the extremes of nutrient/oxygen supply. We also separately studied the basal placenta, site of hormoneproduction and the placental labyrinth, site of feto-maternal exchange.Methods: Pregnant rats were provided either an ad libitum (AdLib) diet orwere restricted to 50% of the food of ad libitum-fed animals (MFR)beginning at E10. At E20 rat dams were euthanized and gestational sacsfrom left and right mid- and proximal horns dissected. AF volume wasquantified as the difference in sac weight before and after drainage and AFosmolality determined by freezing point depression. The placenta andfetus were separated and weighed. Six placentas from right mid- andproximal horns were separated into basal and labyrinth zones andanalyzed for AQP1 and AQP8 protein expression (Western blot).Results: MFR fetuses from mid- and proximal horns weighed significantlyless than their respective AdLib. Additionally, MFR mid- and proximal hornplacentas were significantly smaller in the basal and labyrinth zones. AFVwas significantly decreased and osmolality was significantly increased in theMFR as compared to AdLib from both mid- and proximal horn sacs. In MFR,AQP1 protein decreased significantly in the placental basal zone from mid-horn placentas and increased significantly in the labyrinth zone from bothmid- and proximal horn placentas. AQP8 was increased in MFR placentalbasal and labyrinth zones from both mid- and proximal horn placentas.Conclusion: The differential effects of MFR on AQP1 and AQP8 suggest thataltered AQPs expression may contribute to reduced AFV and increased AFosmolality.Keywords: AQP, IUGR, placenta, transport

[P12.06].THE EFFECT OF REDUCED SYSTEM BETA TRANSPORTER ACTIVITY ONCYTOTROPHOBLAST CELL DIFFERENTIATION IN VITRO

L Parsons, SL Greenwood, M Westwood, M Desforges*. University ofManchester, United Kingdom

Trophoblast cell turnover maintains a functional syncytiotrophoblast,capable of sufficient nutrient transfer from mother to fetus. Fetal growthrestriction (FGR) is associated with aberrant trophoblast cell turnover1 andreduced activity of certain amino acid transporters, including systembeta2. The system beta substrate, taurine, has a role in cell volume regu-lation3. In other tissues, cellular hydration state influences proliferation,differentiation, apoptosis, and hormone secretion4. We thereforehypothesise system b-mediated taurine transport is important fortrophoblast cell turnover. The objective of this study was to investigatecytotrophoblast cell differentiation following knockdown of system beta(TauT) using siRNA technology.Methods: Cytotrophoblast cells isolated from human term placenta weremaintained in primary culture for 66 hours. Cells were transfected withsiRNA at 18 hours of culture as described previously5. Knockdown of TauTmRNA (SLC6A6) was confirmed using QPCR and effects on system betaactivity was determined by measuring sodium-dependent 3H-taurineuptake. Immunofluorescent staining of desmosomes (allowing visual-isation of multinucleation) and measurement of hCG secretion were usedto assess morphological and biochemical differentiation respectively incells with TauT knockdown compared to untransfected control cells.Results: Transfection of cytotrophoblast cells with 50nM TauT-specific siRNAsignificantly reduced SLC6A6 mRNA expression and system beta activity(p<0.05 Wilcoxon signed rank, n¼5) whereas transfection with non-tar-geting siRNA, included as a negative control, had no significant effect. Mul-tinucleation was reduced by 20-60% (mean:37%, n¼4) in cytotrophoblastcells with TauT knockdown compared to untransfected controls, howeverthere was no effect on hCG secretion (p¼0.6, Wilcoxon matched pairs, n¼5).Conclusion: Morphological, but not biochemical, differentiation of cyto-trophoblast cells is compromised when system beta activity is reduced.This suggests that, as well as being directly important for fetal develop-ment, placental taurine transport is important for trophoblast cell turn-over and normal placental development.References1. Crocker et al (2004). J Pathol 204: 11-18.2. Norberg et al (1998). Pediatr Res 44: 233-238.3. Shennan DB (2008). Cell Physiol Biochem 21: 15-28.4. Lang et al (1998). Physiol Rev 78: 247-306.5. Forbes et al (2009). Placenta 30: 124-129.Funded by The Wellcome Trust.Keywords: taurine, syncytiotrophoblast, fetal growth restriction, placenta


[P12.07].MOLECULAR EXPRESSION OF NHE-3 IN HUMAN PREECLAMPTICPLACENTAS

V Dietrich*1, A Reca1, M Castro-Parodi1, B Maskin2, AE Damiano1.1Laboratorio de Biologıa de la Reproduccion, Catedra de Biologıa Celular,Facultad de Farmacia y Bioquımica. Universidad de Buenos Aires,Argentina, 2Hospital Nacional ‘‘A. Posadas’’, Buenos Aires, Argentina

The exchange barrier between maternal and fetal circulation in thehuman placenta consists, essentially, of two cellular layers, the syncytio-trophoblast (hST), and the fetal capillary endothelium. The hST, a multi-nucleated true syncytium, is the transporting epithelium of the placenta.Transport activity of the hST is essential for a supply of a range of solutesrequired for fetal growth, as well as homeostasis of the cell itself. Thesodium hydrogen exchanger isoform 3 (NHE-3) plays an important role inelectrolyte and water homeostasis. These functions are compromised inpregnancies complicated with preeclampsia. At present it is not knownwhether NHE-3 expression is altered during preeclampsia.We investigated the molecular expression of the NHE-3 in hST termplacentas obtained from uncomplicated (n¼6) and preeclamptic preg-nancies (n¼6).RT-PCR and Western blot assays showed that the expression of NHE-3 wassignificantly reduced in hST from preeclamptic placentas. In normalplacentas NHE-3 was localized in the apical and basal membranes and inthe cytoplasm. However, in preeclamptic placentas NHE-3 was almostundetectable.Further studies are needed to determine whether the diminishedexpression of NHE-3 in preeclamptic placentas may compromise placentalfunction and may contribute to the development of this syndrome.Keywords: NHE-3, syncytiotrophoblast, preeclampsia, human placenta

[P12.08].ENDOSOMAL Ph REGULATION IN POLARIZED BeWo CELLS

I. Ellinger*, R. Fuchs. Medical University Vienna, Austria

Introduction: IgG transcytosis and apical IgG recycling in polarized BeWocells are mediated by hFcRn. Following apical uptake by a fluid phasemechanism, IgG enters apical early endosomes (AEE) from where it ispredominantly recycled (40-50%), either directly or via transferrin receptor-positive recycling endosomes, as well as transcytosed (30-35%). High-affinityFcRn-IgG interaction is pH-dependent and requires mildly acidic endosomalpH (<6.5). As the pH of distinct endosomal subpopulations (AEE and baso-lateral early endosomes (BEE), late endosomes (LE)/lysosomes, recyclingendosomes (RE)) in BeWo cells is completely unknown, but expected to vary,this was analysed using dextran (fluid phase marker) and transferrin.Methods: Endosomal subcompartments were marked with FITC (pH-dependent) and Cy5 (pH-independent)-conjugated dextran or transferrin.Single-organelle flow analysis (SOFA) was applied to determine the pH oflabelled endosomes.Results: Following pulse-chase (15/5min) internalization from apical orbasolateral side, the fluid-phase marker dextran was delivered to peri-nuclear endosomes (common LE) with an average pH of 5.6-5.7. Wheninternalized from the basolateral side for 5 min, dextran entered periph-eral endosomes that exhibited an average pH of 6.7 (BEE). In contrast,following apical internalization for 5 min, dextran-marked endosomes hadan average pH of 7.3 (AEE). This difference in pH among AEE and BEE mightbe explained by a larger fraction of apically internalized fluid phasemarkers entering rapid recycling through more neutral compartments.Supportive to this concept, apically applied transferrin passed peripheralAEE and was recycled via perinuclear recycling compartments (RE). Allendosomes labelled continuously for 20 min exhibited an average pH of6.1; in subsequent pulse/chase experiments transferrin-labelled early (5min) and late (20 min) endosomes exhibited low pH (below 6.0). Thissupports the existence of a sufficiently low pH in AEE to allow for IgG-FcRninteraction. DISCUSSION: Our data for the first time addressed pH-regu-lation in endosomal subpopulations of BeWo cells.Keywords: IgG, hFcRn, endosomal pH, BeWo

[P12.09].CALCIUM AND LIPIDS ASSISTED TRANSPORT OF LEAD IN PLACENTA

J. Foltinova*1, V. Foltin2, E. Neu3. 1Comenius University, Slovakia, 2SlovakTechnical University, Slovakia, 3Umweltmedizin Institut, Germany

Introduction: In recent years great attention has been paid to effect ofheavy metals on the human organism from various points of view.Particularly, relations between lead, placenta and fetus are of specialimportance, since they are connected with the rise of hyperkineticsyndrome (ADHD) in children.Material and Methods: In this work we prepared and evaluated sectionsfrom excisions of placentas of 104 healthy patients. We carried out proof ofCa by the method after Koss, proof of Pb by the new methodical approachafter Foltinova [1] and proof of lipids by histochemical methods. Forevaluation we used light microscope Reichart Polyvar (Germany), scanningelectron microscope PHILLIPS CM (Holland), infrared spectrostrometerSPECORD M80, Carl Zeiss, Jena (Germany) and JEOL JXA 840A (Japan) EDAXelectron probe micro-analyzer.Results: Our results show that: a) calcium and lipids play a role of leadcarriers, b) lead transport through placenta participates in the rise ofhyperkinetic syndrome, c) expelling of iron from haemoglobin by leadleads to insufficient transport of oxygen through transplacental barrier, d)finding lead in placenta may help in preventing this disease that hasincreasing occurrence in the world. Histochemical results were confrontedwith the results of EDAX analysis and infrared spectroscopy.Conclusions: Significant occurrence of lipids in syncytiotrophoblast andinside the villi of placenta in the places of lead occurrence shows that alsolipids are carrier of lead in placenta. This explains why in case of hyper-kinetic syndrome in children lead is cumulated in the striatum of brainwhich contains myelin in neurons. Finding of lead in placenta andumbilical cord blood can be utilized for prevention of the hyperkineticsyndrome. Relevance of this finding has the same value as the relevance offinding IgE for revealing allergic terrain of the newly born child.Reference[1] J. Foltinova, V. Foltin, E. Neu: Occurrence of lead in placenta – importantinformation for prenatal and postnatal development of child. Neuro-endocrin. Letters 28 (2007) 335-340.Keywords: lead in environment, transport of lead in placenta, hyperkineticsyndrome


[P12.10].PLACENTAL EXPRESSION OF INSULIN-LIKE GROWTH FACTORS (IGFs),SYSTEM A AMINO ACID AND GLUCOSE TRANSPORTERS AREDIFFERENTIALLY REGULATED BY PREGNANCY AND NUTRITION IN THEGUINEA PIG

PA Grant*1, KL Kind1, A Sohlstom2, CT Roberts1, JA Owens1. 1University ofAdelaide, Australia, 2Linkoping University, Sweden

The placenta is a major determinant of fetal growth and developmentthrough the delivery of substrates and secretion of hormones. Maternalplasma IGFs increase during pregnancy and promote placental transportand nutrient partitioning near term. The effect of maternal food restrictionin the guinea pig, on placental expression of IGFs, glucose transportersSlc2a1, Slc2a3 and system A amino acid transporters Slc38a2 and Slc38a4,fetal and placenta weight were examined at D30 and D60 of gestation(term w70 days). Guinea pigs were fed either ad libitum (AL) D30 (n¼10),D60 (n¼6) or restricted (R) D30 (n¼10), D60 (n¼10) (70% AL from 2 weeksbefore pregnancy to D34, then 90% AL to D60). Placental Slc2a1, Slc2a3,SLC38a2 and Slc38a4 mRNA were quantitated by real-time PCR.Placental Slc2a1 mRNA in AL(14x) and R(21x) (p<0.0001), IGF-I mRNA inAL(78x) and R(48x) and IGF-II mRNA expression in AL(95x) and R(85x)(p<0.0001) were decreased at D60 compared with D30 regardless ofnutrition. Slc38a2 mRNA (3x) (p<0.05) was increased at D60 compared toD30 in AL fed animals. Feed restriction reduced Slc38a2 mRNA (2x)(p<0.05) at D60.Placental weight correlated negatively with placental Slc2a3 (p<0.01) andpositively with Slc38a4 (p<0.01) in late pregnancy in AL animals. Placentaland fetal weight were correlated positively with placental IGF-II mRNA(p<0.05) and Slc38a4 mRNA (p<0.01), respectively in late pregnancy in Ranimals. Placental weight correlated positively with placental Slc2a1mRNA (p<0.05) and negatively with Slc38a2 mRNA (p<0.02) in earlypregnancy in AL animals.This shows there are opposing gestational changes in expression of IGFsand glucose transporters compared to amino acid transporters in theplacenta and that their associations with placental weight change withincreasing gestation.Keywords: Insulin-like Growth factors, Glucose transporters, System Aamino acid transporters, Nutrition

[P12.11].CORRELATION BETWEEN hCG, cAMP AND AQP9 IN HUMAN PLACENTA

G Marino*1, M Castro-Parodi1, E Zotta2, AE Damiano1. 1Laboratorio deBiologıa de la Reproduccion, Catedra de Biologıa Celular, Facultad deFarmacia y Bioquımica. Universidad de Buenos Aires., Argentina, 2Labo-ratorio de Fisiopatogenia, Departamento de Fisiologıa, Facultad deMedicina, Universidad de Buenos Aires., Argentina

Since trophoblastic abnormalities play a central role in the pathophysi-ology of preeclampsia, it is conceivable that some placental hormoneprofiles such as an increased secretion of human chorionic gonadotropin(hCG) are modified in the maternal circulation, which could affect theplacental function. We have described in preeclamptic pregnancies thatthere are elevated levels of serum hCG with an increased aquaporin-9(AQP9) expression in the syncytiotrophoblast (hST).Several reports indicate that AQP9 expression may be mediated by somefactors induced by cAMP. In addition hCG signal transduction usuallyinvolves cAMP signaling.Here, we are focused on the hypothesis that there is a correlation betweenthe increased levels of hCG and AQP9 expression in preeclampticplacentas, involving a cAMP dependent pathway.Normal placental explants were cultured at different times with 20 mIU/mlof recombinant hCG and 100-500 mM 8-bromo-cAMP (8-Br-cAMP),a potent cAMP analogue. Semiquantitative Western blot and immunohis-tochemistry were performed to evaluate AQP9 expression and localization.In Western blots we observed a stimulatory effect on AQP9 protein expressionafter hCG treatment. AQP9 was localized in the apical membrane of hSTand inthe cytoplasm, and similar results were obtained with placental explantsincubated with 8-Br –cAMP, this effect being time and dose dependent.Our results suggest that hCG may be implicated in the regulation of AQP9expression involving a cAMP dependent pathway in hST.Keywords: Aquaporin 9, hCG, syncytiotrophoblast, cAMP

[P12.12].THE DISTRIBUTION OF CAVEOLAE AND CAVEOLIN-1 IN THE MOUSEPLACENTA AND YOLK SAC

S. Mohanty, C.L. Anderson, J.M. Robinson*. The Ohio State University,United States

Introduction: Caveolae are specialized microdomains in the plasmamembrane and are found in several cell types. Caveolae are characterizedbiochemically by the presence of members of the caveolin protein family.We have studied the mouse placenta and yolk sac to determine ifcaveolin-1 is expressed in these tissues and if so what cell types expressthis protein.Methods: The methods employed in this study were immunoblotting oftissue lysates and immunofluorescence microscopy of cryostat sections ofintact tissue to determine if caveolin-1 is expressed and which cell typescontain this protein. Additionally, we have employed electron microscopyof plastic-embedded tissue samples to study the distribution of caveolae.Results: Immunoblot analysis showed that both placental and yolk saclysates contain caveolin-1. Immunofluorescence analysis showed thatendothelium, smooth muscle cells, and mesothelium contain caveolin-1 inyolk sac but endoderm lacks this protein. The labyrinth of the mouseplacenta is enriched in caveolin-1. However, the distribution of this proteinis restricted to endothelial cells. Caveolin-1 was not detected in any of thethree trophoblast layers. Yolk sac and placenta were also examined byelectron microscopy. Ultrastructural analysis showed morphologically-defined caveolae were present in both yolk sac and placenta. The cellstypes expressing caveolae were the same cells as those identified as beingpositive for caveolin-1 in the immunofluorescence experiments.Discussion: We have identified the cells types expressing caveolae andcaveolin-1 in mouse yolk sac and placenta. Morphologically-defined cav-eolae were present only in cell types expressing the protein caveolin-1.These data on caveolin-1 and caveolae in the placental labyrinth parallelthose from the human placenta since trophoblasts are negative whileendothelial cells are positive.Keywords: Caveolae, Caveolin-1, Yolk sac, Placenta


[P12.13].MODEL FOR OXYGEN TRANSPORT IN THE HUMAN PLACENTA

JS Gill*1, DS Grebenkov2, CM Salafia3,4, DD Vvendensky1. 1Imperial College,United Kingdom, 2Ecole Polytechnique, France, 3Placental Analytics LLC,United States, 4Institute for Basic Research, United States

Goal/Background: The mature placenta is a complex vascular networkextending ultimately to the capillary beds of the terminal villi, the sites ofall oxygen and nutrient exchange between the mother and fetus. Respi-ratory transfer across the placenta to the fetus occurs in three steps: (i)maternal blood bathes the chorionic villi in oxygen, (ii) oxygen permeatesthe villus surface and diffuses into the fetal capillaries, and (iii) oxygen istransported to the fetus via the fetal bloodstream.Materials/Methods: Step (ii) has been modelled by the diffusion of oxygenfrom the villous membrane and the fetal capillaries. The stationaryconcentration c(x,t) of oxygen within each of the villi is the solution of thetwo-dimensional Laplace’s equation, Oc¼ 0, with a fixed concentration cv

at the villous surface and a Robin boundary condition at the capillaryboundary: Dvc/vn ¼ Kc, where v/vn is outward normal derivative, D is theoxygen diffusion constant and K is the permeability of the capillary. Theseequations are solved in regions [panels (d,e,f)] determined by the villusand capillary boundaries obtained from digitized images [panels (a,b,c)].Results: The solution for the oxygen concentration determines the diffu-sive current of oxygen across the capillaries. Many factors are expected toinfluence this current, including the numbers and shapes of villi andcapillaries. Our initial analysis for the sections below indicates that panel(c) yields the largest flux per unit perimeter length and area of the villi,followed by panel (b) and then panel (a).Conclusions: The geometrical shapes and spatial distributions of the villiand capillaries are important placental characteristics for the transport ofoxygen to the fetus. Once the main factors that determine oxygen trans-port have been precisely identified, this approach, applied to digitizedplacental slides that allow analysis of many hundreds of villi per slide (andmultiple slides per placenta) should provide a quantitative basis fordiscriminating between normal and abnormal.

Keywords: diffusion, oxygen flux, placental capillaries, terminal villi

[P12.14].INSULIN INCREASES EXPRESSION AND ACTIVITY OF EQUILIBRATIVENUCLEOSIDE TRANSPORTER 2 IN HUMAN PLACENTA MICROVASCULARENDOTHELIAL CELLS FROM GESTATIONAL DIABETES

Carlos Salomon*, Francisco Westermeier, Paola Casanello, Luis Sobrevia.Pontificia Universidad Catolica de Chile, Chile

Human endothelial cells from placenta microcirculation (hPMEC) expressfunctional equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2)in culture. ENTs-mediated adenosine transport is regulated by insulin inhuman umbilical vein endothelium from normal or gestational diabetic(GD) pregnancies, but no information is available regarding the effect ofthis hormone in hPMEC. We studied insulin effect on hENT2 expressionand activity in hPMEC from GD pregnancies.Methods: Primary cultures of hPMEC (passage 2) from normal or GDpregnancies were cultured under standard conditions. hENT2-mediatedadenosine transport (0-500 mM adenosine, 4 mCi/ml [3H]adenosine, 22�C),hENT2 protein abundance and mRNA level were determined.Results: Insulin increased hENT2 mediated adenosine transport by w1.5-fold, but only w1.2-fold in hPMEC from normal compared with GD preg-nancies, respectively. Basal expression of hENT2 (protein and mRNA) was notsignificant different in cells from normal or GD pregnancies. Insulinincreased in a concentration-dependent manner hENT2 protein abundance(half-maximal effect (SC50) w1 nM), mRNA level (SC50 w1.2 nM) andtransport activity (SC50 w1.1 nM) in hPMEC. However, all insulin effects werehigher (w1.4-fold) in cells from normal compared with GD pregnancies.Conclusion. Human placenta microvascular endothelium is less respon-sive to insulin regarding hENT2 expression and activity. This phenomenoncould partially explain the insulin resistance exhibited by some fetusesfrom gestational diabetes.Supported by FONDECYT 1070865/1080534. C.S. holds a Faculty ofMedicine, PUC-PhD fellowship. F.W. holds a CONICYT-PhD fellowship.Keywords: Adenosine, Gestational Diabetes, hPMEC, ENTs


[P12.15].HIGH D-GLUCOSE INHIBITS EQUILIBRATIVE NUCLEOSIDETRANSPORTER 1 (hENT1) ACTIVITY AND EXPRESSION BY ACTIVATIONOF TYPE II RECEPTOR FOR TRANSFORMING GROWTH FACTOR ß1(TGF-ß1) IN HUMAN FETAL ENDOTHELIUM

Jose Luis Vega*, Carlos Puebla, Paola Casanello, Luis Sobrevia. Cellular andMolecular Physiology Laboratory (CMPL) and Perinatology ResearchLaboratory (PRL), Department of Obstetric and Gynaecology, MedicalResearch Centre (CIM), School of Medicine, Pontificia U, Chile

Introduction: The transforming growth factor ß1 (TGF-ß1) inhibitsexpression and activity of hENT1 in a nitric oxide (NO)-dependent mannerin human umbilical vein endothelial cells (HUVEC). High extracellular D-glucose (25 mM) also increase the release of TGF-ß1 from primary culturesof HUVEC leading to altered transport of L-arginine and NO synthesis, mostlikely due to activation of type II TGF-ß1 receptors (TßRII). We have studiedwhether TGF-ß1 is involved in the inhibitory effect of high D-glucose onhENT1 activity and expression in HUVEC.Methods: Cells were exposed to D-glucose (5-25 mM, 6-24 h) and/or TGF-ß1 (2 ng/ml, 6 h) and [3H]adenosine transport (4 mCi/ml, 20 sec, 22oC) wasmeasure in absence or presence S-(4-nitrobenzyl)-6-thio-ionosine (ENT1inhibitor). hENT1 mRNA was estimated by semi-quantitative PCR andprotein level by western blot. The TGF-ß1 or high D-glucose effect onreporter activity of plasmid constructs containing a promoter region ofSLC29A1 (-1114 bp to ATG, pGL3-hENT1-1114) was also analyzed. All assayswere performed in cells transduced with an adenovirus to induceexpression of a truncated form of TßRII (Ad-tTßRII) or with a controladenovirus (Ad-control, empty vector).Results: D-glucose reduced in a dose-dependent manner adenosinetransport via hENT1, mRNA expression, protein abundance, and pGL3-hENT1-1114 transcriptional activity. TGF-ß1 effects were absent in cellstransduced with Ad-tTßRII.Conclusion: High D-glucose reduced hENT1 activity and expressionrequires activation of native type II receptor for TGF-ß1 in primary culturesof HUVEC.FONDECYT 1070865/1080534, CONICYT AT-24090199 (Chile). JL Vega andC Puebla hold CONICYT-PhD fellowships.Keywords: High D-glucose, hENT1 , TGF-ß1, Human Fetal Endothelium

[P12.16].INSULIN RECEPTOR ISOFORMS -11 (IR-A) AND +11 (IR-B) AREEXPRESSED AND COULD MEDIATE INSULIN-INCREASED EXPRESSIONAND ACTIVITY OF THE EQUILIBRATIVE NUCLEOSIDE TRANSPORTERTYPE 2 IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS FROMPREGNANCIES WITH GESTATIONAL DIABETES

Francisco Westermeier*, Carlos Salomon, Paola Casanello, Luis Sobrevia.Pontificia Universidad Catolica, Chile

Adenosine is an endogenous nucleoside that is taken up by the humanumbilical vein endothelial cells (HUVEC) via equilibrative nucleosidetransporters (predominantly hENT1 and hENT2). Both the expression andactivity of hENT1 are decreased in HUVEC from gestational diabetes (GD),as well as in HUVEC exposed to insulin. However, insulin increases aden-osine transport in HUVEC from normal pregnancies and regulates SLC29A2(for hENT2) gene expression. We studied insulin effect on adenosinetransport mediated by hENT2 in HUVEC from pregnancies with GD.Methods: HUVEC from normal and GD were exposed (8 hours) to insulin(0.1-10 nM). [3H]Adenosine transport (4 mCi/ml, 20 s, 22�C) was measuredin absence or presence of nitrobenzylthioinosine (NBTI, 0.01-10 mM, 30min). SLC29A2 transcriptional activity was estimated by firefly and renillaluciferase activity from two upstream sequences (1500 and 600 bp fromthe ATG) subcloned into pGL3-basic vector. Insulin receptors isoforms -11(IR-A) and +11 (IR-B) mRNA level were determined.Results: Insulin increases hENT2-adenosine transport (SC50 w0.53 andw0.51 nM), protein abundance (SC50 w0.78 and w0.82 nM) and mRNAlevel (SC50 w3 and w3 nM) were increased by insulin in cells from normaland GD pregnancies, respectively. pGL3-hENT2-1500 and pGL3-hENT2-600

luciferase promoter activity was similar in cells from normal and GDpregnancies. Only the pGL3-hENT2-1500 promoter activity was increasedby insulin in a similar pattern in cells from normal and GD pregnancies. IR-A mRNA level was higher (w1.3-fold, P<0.05) in GD compared withnormal pregnancies. However, IR-B mRNA level was similar in bothconditions. IR-A was higher that IR-B mRNA levels in normal (w1.5-fold)and GD (w1.8-fold) pregnancies.Conclusion: Insulin could increase adenosine transport via hENT2 byincreasing expression of this transporter isoform in HUVEC from GD andnormal pregnancies. In addition, GD is a pathological condition where theplacenta/fetal tissue-predominat isoform IR-A, could play a critical role ininsulin signalling.Supported by FONDECYT 1070865/1080534 (Chile). F. Westermeier holdsa CONICYT-PhD fellowship. C. Salomon holds a Faculty of Medicine,PUC-PhD fellowship.Keywords: Gestational Diabetes, Insulin receptor, Human equilibrativenucleoside transporter, Adenosine


[P13.01].SINGLE NUCLEOTIDE POLYMORPHISMS IN ANGIOGENESIS REGULATINGGENES ARE ASSOCIATED WITH PREGNANCY COMPLICATIONS

PH Andraweera*, SD Thompson, RC Novak, VJ Zhang, GA Dekker, CTRoberts. University of Adelaide, Australia

Introduction: Angiogenesis is crucial for normal placentation and whenderanged is implicated in pregnancy complications. Vascular endothelialgrowth factor (VEGF) is a potent pro-angiogenic factor and thrombo-spondin1 (THBS1) an anti-angiogenic factor. We aimed to determinewhether functional polymorphisms in VEGF (VEGF C2578A and VEGFC936T) and THBS1 (THBS1 A2099G) genes are associated with pregnancycomplications.Methods: 1380 nulliparous pregnant women and their partners wererecruited prospectively at the Lyell McEwin Hospital and women moni-tored throughout pregnancy. Gestational hypertension (GHT),Preeclampsia (PE), Small for gestational age (SGA) and PE+SGA pregnan-cies were classified using strict guidelines. Uncomplicated pregnancieswere selected as controls. Peripheral blood was collected from couples andcord blood collected at delivery. DNA extraction from buffy coats andgenotyping were performed at the Australian Genome Research Facilityusing the Sequenom MassARRAY system. To date the first 700 mother,father, baby trios have been genotyped. Genotyping results for GHT(n¼58), SGA (n¼53), PE (n¼37), PE+SGA (n¼11) were compared withcontrols (n¼250). Data were analysed using ANOVA and Chi Square.Likelihood Ratios (LR) were calculated.Results: Neonatal VEGF C2578A was associated with PE (p¼0.007, LR¼10.3) and neonatal VEGF C936T with SGA (p¼0.029, LR¼6.3). MaternalTHBS1 A2099G was associated with GHT (p¼0.027, LR¼6.0), paternalTHBS1 A2099G was associated with PE+SGA (p¼0.036, LR¼6.3) andneonatal THBS1 A2099G was associated with SGA (p¼0.038, LR¼6.2).Neonates of fathers with THBS1 A2099G GG genotype were 701g lighter(p¼0.026) and 4.8cm shorter (p¼0.001) than those of GA and 785g lighter(p¼0.008) and 4.9cm shorter (p¼0.001) than those of AA. Maternal VEGFC2578A polymorphism was associated with the urinary protein:creatinineratio.Conclusion: Our results suggest that VEGF C2578A, VEGF C936T and THBS1A2099G gene polymorphisms are associated with pregnancy complica-tions. Ongoing research will determine the role of these polymorphisms inplacental angiogenesis and function.Keywords: angiogenesis, vascular endothelial growth factor, thrombo-spondin1, polymorphisms

[P13.02].PREDICTION OF PREECLAMPSIA WITH ELEVATION IN ERYTHROBLASTCOUNT IN MATERNAL BLOOD

Fatemeh.* MD Davari Tanha*, Jalleh Mohammad pour, MD , Mahbod Kaveh.Tehran University Medical Sciences, Republic of Islamic, Iran

Introduction: The predominant etiologic theory of preeclampsia is thatreduced uteroplacental perfusion is the unique pathogenic process in thedevelopment of preeclampsia. Maternal and fetal erythroblast counts areelevated in the peripheral blood of pregnant women with preeclampsia.The purpose of this study was to examine whether this elevation actuallyoccurs before the clinical onset of the disorder.Study Design: In a prospective cohort survey erythroblasts wereenumerated in 599 maternal blood samples obtained in 19-26 weeks withsingleton pregnancy.After complete blood count a peripheral blood smearwas done and erythroblast was counted , and results were subsequentlycorrelated with pregnancy outcomes. The data were analyzed by SPSS13.0.Independent sample t-test and Fisher’s exact test was used. A p valueof <0.05 was considered statistically significant.Results: Significantly higher quantities of erythroblasts (mean 2.46�1.23 vs0.44�0.55;p¼0.009)were detected in blood samples obtained from womenwho later acquired preeclampsia (n¼50) than in blood samples from thecontrol Cohort (n¼549). Intrauterine growth restriction was accompany bya similar rise in erythroblast count(mean NRBC 0.82�0.8 in preeclampticgroup vs 0.59�0.85 in normotensive group;p¼0.009). Mean gestational agewas less in preeclamptic group(37.58�1.45 vs 39.07�0.94, p¼0.009). Onthe basis of 1.5 erythroblast as point of convergence there was sensitivity¼61.45, specificity¼93.02, NPV¼98.16, accuracy¼91.65Conclusion: Because a large proportion of the erythroblasts in maternalblood are fetal origin, our data suggest that fetal-maternal cell traffic isaffected early in pregnancies that are later complicated by preeclampsia.Keywords: erythroblasts, preeclampsia, IUGR, fetal cell traffic


[P13.04].PLASMINOGEN, THE PLACENTA AND PREGNANCY OUTCOME

RC Nowak*, SD Thompson, J Zhang, GA Dekker, CR Roberts. Research Centrefor Reproductive Health, Australia

Introduction: Plasminogen (PLG) has been shown to play a crucial role inplacental development. Poor placental development is associated withseveral potentially life-threatening pregnancy complications, includingpreeclampsia (PE) and small-for-gestational-age (SGA) babies.Aim: To determine if two single nucleotide polymorphisms (SNP) withinPLG, rs28598789 and rs425114, are associated with pregnancy outcome.Method: First trimester placental tissue was collected from womenundergoing termination of pregnancy to determine PLG expression inplacental villous tissue. To examine genotype with pregnancy outcome,blood was collected prospectively from pregnant women at 15 weeksgestation, their partners and neonates at birth. DNA, extracted from blood,was genotyped by the Australian Genome Research Facility with the use ofSequenom MassArray technology. Pregnancies were fully characterised andclassified into control, PE or SGA after delivery by an experienced obste-trician. Detailed clinical information was collected from each participant.Results: PLG is expressed in placenta throughout first trimester , with nodifference in mRNA levels across 6-12 weeks gestation or between sexes.In women, the frequency of the PLG rs28597879 TT genotype wasincreased in those who developed PE (n¼41) compared to controls(n¼285, p¼0.011, LR 6.43 p¼0.016). This genotype was also positivelyassociated with the maternal birthweight (p¼0.024). Likewise there wasan increase in the frequency of the rs425114 C-allele in women (n¼41,p¼0.007 LR 7.9) and men (n¼30, p¼0.019 LR 6.2) who parented a PEpregnancy compared to controls (n¼282 and n¼240 respectively). Thissame increase was also observed in neonates who were SGA (n¼51,p¼0.014 LR 6.2) compared to controls (n¼241).Conclusion: This is the first study to show an association between thesepolymorphisms of PLG and pregnancy outcome. PLG is expressed in placentaduring first trimester and functional SNPs within PLG may be associated withaltered placental development and subsequent pregnancy outcome.Keywords: Genotype, Plasmiogen, Pregnancy outcome

[P13.06].EXPRESSION OF ANGIOGENIC FACTORS IN PREECLAMPSIA

Ji Kwon Park*, Jong Chul Baek, Jeong Kyu Shin, Won Jun Choi, Soon Ae Lee,Jong Hak Lee. Gyeongsang National University, Republic of Korea

Elevated expression of soluble vascular endothelial growth factor receptor-1 (sFlt-1), an antiangiogenic protein, in preeclampsia plays a major role inthe pathogenesis of this serious disorder of human pregnancy. Althoughreduced placental oxygenation is thought to be involved in the patho-genesis of preeclampsia, it is unclear how oxygen regulates placental sFlt-1expression. The aims herein were to investigate sFlt-1 expression in in vivoand in vitro physiological and pathological models of human placentalhypoxia and to understand the role of phosphatidylinositol-3-kinase(PI3K) pathway in regulating the expression of this molecule.I would like to examine placental sFlt-1 and VEGF expression inpreeclamptic pregnancies (in vivo pathological hypoxia) and models ofplacental hypoxia (Human choriocarcinoma trophoblast cells with hypoxia-inducing chemical agents). Additionally, using models of placental hypoxia,we have investigated the mechanisms by which reduced oxygenation (invitro hypoxia) regulate sFlt-1 expression. Finally, we have determinedwhether PI3K pathway, in particular PI3K, Akt, HIF-1, plays a direct role inregulating the expression of sFlt-1 in the models of placental hypoxia.Through this study, exploration of some agents that neutralize the excesssFlt-1 may also be promising therapeutic modalities in patients withpreeclampsia.Keywords: Preeclampsia, Human choriocarcinoma trophoblast cells,sFlt, PI3K


[P13.08].COLLAGEN INTEGRITY OF THE UTERINE CERVIX CORRELATES WITHAMNIOTIC FLUID CYTOKINE LEVELS, PROVIDING A NOVEL METHODTO DETECT INTRA-AMNIOTIC INFLAMMATION

SM Keeler*1, O Rust2, E Bornstein1, M Sottile3, CM Salafia4. 1New YorkUniversity, United States, 2Lehigh Valley Hospital, United States, 3Univer-sity of Oregon, United States, 4Placental Analytics LLC, United States

Objective: To investigate the correlation between the histological stainingcharacteristics of cervical collagen and amniotic fluid cytokines in womenwith midtrimester short cervix.Study Design: Amniotic fluid (AF) and cervical biopsy samples wereobtained from women with a midtrimester transvaginal cervical length.25mm. AF cytokine concentrations were quantified. From a routine stainedslide, one 40x photomicrograph was selected from each specimen by anobserver blinded to the amniotic fluid cytokine levels. The field was selectedto be at the apparent mid point of the biopsy and to fill the 40x field of view.The photomicrographs were analyzed using RGB channels; the G and Bchannels were discarded due to the predominance of R channel variancegiven the normal eosinophilia of collagen. The red channel histogram wasextracted and aspects of its distribution, including mean, standard devia-tion, skew and kurtosis, were calculated. Since these variable wouldreasonably be intercorrelated, a single collagen staining factor score (CFS)was extracted from these data by principal components analysis andcompared to the cytokine concentrations using Spearmans correlation. Ahigh score represents impaired cervical integrity with the biopsy containingloose, poorly interconnected collagen fibrils and has an increased amount ofwhite space in the image. A low score represents normal dense, closelyinterconnected collagen fibrils, with very little white space in the image.Results: Thirty-three paired AF and cervical biopsy specimens wereanalyzed. A significant association was detected between the collagenscore and AF IL-6 (p¼0.04), IL-8 (p¼0.03), Eotaxin (p¼0.04), IP-10 (p¼0.04)and MCP-1 (p¼0.02) levels.Conclusion: Analysis of collagen integrity on H&E stained slides by a semi-quantitative scoring system had previously proved fruitless in this data set(Salafia CM, unpublished observations). A simple R channel analysis allowsextraction of collagen structural features that highly correlate withinflammatory cytokines, suggesting that intra-amniotic inflammation andthe structural integrity of the cervix are related. This allows an aspect ofcervical integrity to be reliably and reproducibly quantified, that previ-ously could not be assessed on histology slides.

Keywords: short cervix, cervical collagen, cytokines, intra-amnioticinfection

[P13.09].‘‘JELLY-LIKE’’ HETEROGENEOUS PLACENTA AS PREDICTOR OF ADVERSEPREGNANCY OUTCOME. SOME CLINICAL, ULTRASOUND, ANDMORPHOLOGICAL FEATURES

O. Solovyov*1, T. Zadorozhna2, I. Sudoma1,3, Y. Goncharova1. 1ReproductiveMedicine Clinic ‘‘Nadija’’, Ukraine, 2Institute of Pediatrics, Obstetrics andGynecology, Academy of Medical Sciences of Ukraine, Ukraine, 3Nationalmedical Academy of Postgraduate Education named after P.L. Shupyk,Ministry of Health of Ukraine, Ukraine

Objective: to investigate outcome of pregnancies with sonographic find-ings of thick and heterogeneous placenta, comparing histopathological,clinical and Doppler data.Methods: a series of 13 cases were analyzed. A ‘‘jelly-like placenta’’ wasdefined as a thick heterogeneous placenta with a layer of decreasedechogenecity with visible slow swirling movements of contents, whichquivered like jelly to ultrasound probe jerks. Doppler evaluation of theplacenta and uteroplacental circulation was performed. The pregnancycourse and neonatal outcome of present cases were reviewed. Morpho-logical evaluation of placenta after delivery was done.Results: Perinatal death occurred in 5 cases (38,5%). In all these cases theuterine artery Doppler was abnormal or secondary ‘‘normalized’’ afterpreviously increased resistance for uterine arteries blood flow. In 4 cases(30,8%) neonatal outcome was good. In these patients uterine arteriesDoppler was normal during pregnancy. In 4 women with boundary uterineDoppler indices neonatal outcome was satisfactory but with low-birth-weight babies.Morphologically typical vascular changes were noticed, namely plasmor-ragia, strokes and thrombosis, local blood stasis in the vessels of decidua,villi and chorionic lamina, full arteries obliteration of some stem villi of IIand III line. The presence of distally located parts of villi fixed in fibrinoid,pushed aside from intervillous blood flow, narrowing of intervillous spacedue to intervillous strokes, thrombosis, conglomerates of villi fixed byfibrinoid and presence of thin fibres structures with severe edema wereseen.Conclusions: the sonographic presence of thick heterogeneous ‘‘jellylikeplacenta’’ is strongly associated with an adverse pregnancy outcome,especially in case of abnormal uterine artery Doppler or secondary‘‘normalisation’’ after previously abnormal one.Keywords: jellylike, placenta


[P13.10].DYNAMICS OF BIOPYRRINS (BPn) AS A MARKER OF PSYCHOLOGICALSTRESS IN PREGNANT WOMEN ACCORDING TO THE MODE OF DELIVERY

M.S. Suzuki*, R.K. Kawaguchi, T.T. Tanemoto, N.U. Umehara, S.W. Wada,K.O. Ohura, T. Onda, S. Isonishi, K. Ochiai, T. Tanaka. Jikei University Schoolof Medicine, Japan

Objectives: BPn, one of the bilirubin oxidative metabolites, are generatedfrom bilirubin as a result of this scavenging action against free radicals. Thelevel of BPn in urine reflects oxidative stress and has been reported toserve as a marker of psychological stress. We focused on the dynamics andsignificance of BPn in intra-partum women with special attentions to themanner of delivery.Methods: Subjects were composed of full-term pregnant women withoutany complication, and classified into 4 groups : normal vaginal deliverywithout any intervention (ND), delivery with the administration ofoxytocin (OX), cesarean section (CS), and delivery with epidural anesthesiaplus oxytocin (EOx). Spot urinary samples were collected before and afterthe delivery, and the BPn levels were compared.This study was approved by the Institutional Review Boards of The JikeiUniversity, and the Clinical Study in Jikei-Aoto Hospital. And the informedconsent was obtained from all patients registered.Results: Cases of 57(ND), 34 (OX), 37(CS), and 11(EOx) were analyzed. Themean age and gestational week of those were 31.9�5.4 (mean�SD) yearsand 38.5�2.1. No significant difference was found in age, gestational weekand other factors, such as BMI, intra-partum hemorrhage, duration of thedelivery among the group. The BPn levels before the onset of delivery werenot significantly different. Although significant elevation after the deliverywas found in ND-group and OX-group (1.3 and 2.5 fold), no statisticallydifferent was seen between before and after the delivery, in CS- and EOx-group. Moreover, BPn levels in EOx-group were clearly elevated comparedwith other delivery-modes with the significant difference.Conclusion: These data suggested that BPn is also useful marker inpregnancy. Researchs concerning the existence of BPn in placenta and theirclinical applications for predicting obstetrical disorders are in progress.Keywords: biopyrrin, delivery, oxidative stress

[P13.11].STATUS OF ESSENTIAL FATTY ACID, LONG CHAIN POLYUNSATURATEDFATTY ACID AND TRANS FATTY ACID IN MATERNAL AND FETALBIOLOGICAL COMPARTMENTS OF PREGNANT ADOLESCENTS

ORC Oliveira, MG Santana, FC Domingues, FLC Sardinha, GV Veiga, MGTavares do Carmo*. Universidade Federal do Rio de Janeiro, Brazil

Introduction: In Brazil, 21% of the babies born in the public health systemcome from adolescent mothers. Studies of the fatty acids composition ofmaternal and fetal placental tissues have been conducted in adult preg-nant women, but there is a remarkable lack of similar studies in pregnantadolescents. In this study, we investigated the fatty acids composition ofmaternal and fetal placental tissues, as well as in maternal and umbilicalcord plasma, in 30 Brazilian adolescent healthy mothers.Methods: Levels of Trans fatty acids (tFA), essential fatty acids (EFAs),arachidonic (20:4,n-6 AA) acids, eicosapentaenoic (20:5, EPA) and docosa-hexaenoic (22:6, DHA) acids of the n-3 family were measured by gas-liquidchromatography. Results were expressed as a percentage of total fatty acids.Results: We found that the total concentrations of AA, EPA and DHA werehigher in cord than in maternal plasma. In addition, we observeda decrease in AA and EPA concentration in cord plasma compared to fetalplacental tissues. The percentage of EFAs and tFAs were lower in cord thanin maternal plasma. Furthermore, the level of DHA was inversely related tolinoleic acid concentration in fetal placental tissues.Discussion: We found higher accumulation of AA, EPA and DHA in cordplasma. This fact provides evidence of a selective long chain poly-unsaturated fatty acid transfer across the placenta. We speculate that theincreased conversion of AA to the eicosanoids and the DHA transfer to thefetal circulation for neurodevelopment may be responsible for thedecreased concentrations of of these fatty acids in cord plasma comparedto fetal placental tissues. In a future study, we intend to compare theplacental fatty acid transfer in adolescent and adult mothers.Keywords: fatty acids, maternal anda fetal placental, pregnant,adolescents


[P13.12].IGF2 AND IGF2AS POLYMORPHISMS ASSOCIATED WITH ADVERSEPREGNANCY OUTCOMES

S.D. Thompson*1,2, R.C. Nowak1,2, J.V. Zhang1,2, G.A. Dekker1,2, C.T.Roberts1,2. 1Research Centre for Reproductive Health, Australia, 2Disciplineof Obstetrics and Gynaecology, University of Adelaide, Australia

Objectives: IGF-II is an important determinant of trophoblast invasion,differentiation and subsequent placental function and thereby promotesfetal growth. IGF2 antisense (IGF2AS) overlaps IGF2 in the opposite orien-tation and is highly expressed in first trimester chorionic villi. IGF2 andIGF2AS are imprinted and expressed from the paternal allele.Aim: To determine whether maternal, paternal or fetal polymorphisms ofIGF2 and IGF2AS are associated with common pregnancy complications.Methods: 586 nulliparous caucasian couples who required no fertilitytreatment were recruited prospectively from the Lyell McEwin Hospital,Adelaide, Australia. Pregnancy outcomes were classified into healthy(n¼261), preeclampsia (PE, n¼38), gestational hypertension (GH, n¼51),small-for-gestational-age (SGA, n¼60) or preterm birth (PTB, n¼32) by anexperienced obstetrician. Four single nucleotide polymorphisms wereinvestigated, IGF2 rs680 and rs3741204 and IGF2AS rs1004446 andrs1003484. DNA was extracted then genotyped by Australian GenomeResearch Facility using the Sequenom massARRAY System. Data wereanalysed using Chi Square and Odds Ratios (OR).Results: In partners, both IGF2 rs680 and IGF2AS rs1003484 were associ-ated with GH (P<0.00096 OR¼3.10, P<0.0073 OR¼2.58, respectively). Inneonates, IGF2 rs680 was associated with PE (P<0.0061 OR¼3.22), SGA(P<0.01367 OR¼2.35) and PTB (P<0.033 OR¼2.71), while IGF2ASrs1003484 was associated with PTB (P<0.035 OR¼2.77). In mothers IGF2rs680 was associated with PTB (P<0.025, OR¼2.41).Discussion: We are the first to identify associations between paternal andneonatal genotype for paternally expressed genes and adverse pregnancyoutcome. For these paternally expressed genes, a strong association wasseen between paternal genotype and GH, as well as neonatal genotypewith many adverse pregnancy outcomes. These data suggest that neonatalIGF2AS and particularly IGF2 genotype confers a greater risk of developingpregnancy complications, with a 2.4-3.2 fold higher risk of PE, SGA andPTB. Future investigations will identify a functional role in the placenta forthese polymorphisms.Keywords: Association, Polymorphism, IGF2, Genetic

[P14.01].PREDICTORS OF PREGNANCY COMPLICATIONS IN WOMEN WITHSYSTEMIC LUPUS ERYTHEMATOSUS

D Chirico*, I N Bruce, P N Baker, I P Crocker, C L Tower. University ofManchester, United Kingdom

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune diseaseassociated with pregnancy complications (notably pre-eclampsia) in whichdysregulation of CD4+/CD25+/Foxp3+ T-cells (T-regulatory cells) is considereda contributory factor. Transforming Growth Factor beta-1 (TGF-b1) is a cyto-kine involved in both T-reg cell activation and recognised alterations in arterialstiffness, a potential predisposing factor for pre-eclampsia. In this preliminarystudy, we have measured these components in the peripheral circulation ofwomen with SLE and healthy pregnant and non-pregnant individuals.Methods: T-reg cells were assessed by flow cytometry (CD4+/CD25+/Foxp3+), TGFb1 by ELISA and arterial stiffness, expressed as stiffness index(SI), by pulse wave analysis.Results: Preliminary results show that CD4+/CD25+ T-reg cells (asa proportion of total lymphocytes) increased in early pregnancy (12 weeksgestation) [15.4% median (11.8-20.9) interquartile range, N¼9] comparedto non-pregnant healthy controls [8.9% (7.5-9.3), N¼11, p¼0.001]. Simi-larly, Foxp3 positivity of these T-reg cells was also elevated [25.2% (18.2-32.1), N¼6 vs. 12.1% (10.9-15.8), N¼3, p¼0.02]. CD4+/CD25+ T-reg cellswere significantly decreased in SLE early pregnancy [10.43% (8.4-11.1)],compared with healthy early pregnant controls (p¼0.03).TGFb1 was significantly higher in non-pregnant healthy individuals [1.6activation index (1.5-1.9), N¼8] compared with non-pregnant SLE patients[1.0 (0.9-1.2), N¼3, p¼0.02], but there was no difference in early healthypregnancy [1.81 (1.5-2.3), N¼4].SI was non-significantly elevated in women with SLE [9.0m/s (7.2-13.2),N¼6, p¼0.06)] compared to non-pregnant healthy women [7.1m/s (6.7-8.3), N¼9], and no further differences were defined in early pregnancy[7.1m/s (6.5-8.2), N¼8].Discussion: In conclusion, increased levels of CD4+/CD25+/Foxp3+ T-regcells, but not TGFb1, may be a component of uncomplicated pregnancy.Arterial stiffness may not be a predisposing factor for pregnancy compli-cations in SLE, but a failure to acquire necessary T-reg cells may impactupon pregnancy outcome. Work is ongoing to confirm this suggestion.This study was funded by the Wellcome Trust.


[P14.02].THE RELATION BETWEEN THE INCIDENCE OF PLACENTA PREVIAACCRETA AND THE METHODS OF UTERINE CLOSURE AT PREVIOUSCESAREAN SECTION

C Sugiyama*, S Sumigama, Y Mano, T Kotani, H Hayakawa, F Kikkawa.Nagoya University Graduate School of Medicine, Japan

In placenta previa, history of cesarean section (CS) is a risk factor forplacenta accreta. In this study, we carried out to survey the relationshipbetween the two methods of uterine closure (continuous vs interrupted) atprevious CS and the incidence of placenta accreta.We reviewed records ofpatients in 11 tertiary hospitals in Japan between 1994 and 2008. In total 94,968 deliveries, 867 patients had placenta previa, and 119 of whom had oneor more histories of previous CS and 80 records were obtained. Four patientswith classical incision or closure by unabsorbable material were exceptedand 76 patients with lower transverse incision and closure by absorbablematerial were included for this study. In this sample, 31 patients had beendiagnosed as placenta previa with accreta pathologically. 31 patients hadsingle-layer closure include 7 with continuous suture (2 placenta accreta)and 24 with interrupted suture (10 placenta accreta). 45 patients haddouble-layer closure include 17 with continuous suture (12 placenta acre-eta) and 28 with interrupted suture (7 placenta accreta). We record thatplacenta accreta occurred in the patients with single-layer is 39% and 42% ofdouble-layer group, which showed no significant difference (p¼0.76).However, placenta accreta occurred in 52% of the patients with continuousgroup and 26% of interrupted group, which showed a significant differencewith p¼0.034. When limited to double-layer closure, placenta accretaoccurred in 71% of continuous group and in 25% of interrupted group, whichshowed strong difference (p<0.01). In addition, multivariable logisticregression analysis adjusted for confounder (gravid, history of inducedabortion, placental location, number of previous CS) also showed significantdifference (p¼0.023, OR¼8.15).In conclusion, we showed the possibilitythat continuous suture for the closure of uterine incision at previous CScould be a risk factor for accreta in placenta previa with history of CS.Keywords: placenta accreta, cesarean section, uterine incision, suturemethod

[P14.05].DO PREGNANCIES WITH PREECLAMPSIA HAVE SMALLER PLACENTAS? APOPULATION STUDY OF 317 688 PREGNANCIES WITH AND WITHOUTGROWTH RESTRICTION IN THE OFFSPRING

A Eskild*, LJ Vatten. 1Dep. of Obstetrics and Gynecology and MedicalFaculty Division Akershus University Hospital, Norway, 2Inst. of PublicHealth. Norwegian University of Science and Technology, Norway

Background: Preeclampsia is thought to be caused by placental dysfunc-tion. A small placenta may serve as an indicator of dysfunction.Aim: To study whether pregnancies with preeclampsia have smallerplacentas than normotensive pregnancies. This was studied in pregnancieswith and without small for gestational age (SGA) offspring, defined as birthweight lower than the 2.5 percentile.Study population: All singleton births in Norway from 1999 through 2004,a total of 317 688 pregnancies.Outcome measures: Proportion of pregnancies within placental weighttenths (based on z-scores of placental weight).Results: Among pregnancies with SGA offspring, close to 60% were in thelowest tenth of placental weight, with similar proportions of preeclamptic(59.9%) and normotensive pregnancies (61.4%). Less than 1% of pregnancieswith SGA offspring, regardless of preeclampsia status, were in the highesttenth of placental weight. Pregnancies without SGA offspring were evenlydistributed across placental weight tenths, but in these pregnancies, theproportion with preeclampsia was slightly higher both at the lowest (9.5%versus 8.8%) and the highest tenth (11.9% versus 10.2%) of placental weight.Conclusion: We found that pregnancies with and without preeclampsiawere similarly distributed across placental weight tenths, with consistentfindings in pregnancies with and without SGA offspring. Our findingssuggest that a small placenta is not associated with peeclampsia, but withSGA in the offspring.Keywords: Placental weight, Preeclampsia, Fetal growth restriction,Population study


[P14.06].DECREASED ANTIOXIDANT STATUS OF HUMAN CERVICO-VAGINALFLUID (CVF) WITH IMPENDING LABOUR: DEVELOPING A PREDICTIVECAPACITY

HM Georgiou*1,2, YJ Heng1,2, MKW Di Quinzio1,2, M Permezel1,2, GE Rice1,3.1University of Melbourne, Australia, 2Mercy Hospital for Women, Australia,3Baker IDI Heart & Diabetes Institute, Australia

Introduction: Proteomic analysis of CVF by 2D electrophoresis revealedsignificant differential expression of four major antioxidant enzymesduring late pregnancy and labour. Cu,Zn superoxide dismutase (Cu,ZnSOD) and thioredoxin-1 (Trx-1) were significantly decreased in sponta-neous term labour while glutathione-s-transferase and peroxiredoxin-2were significantly increased with impending labour [1]. The differingexpression of these antioxidants prompted the investigation of the totalantioxidant capacity (TAC activity assay) of CVF, both prior to and in termlabour. The study further investigated the temporal changes of Cu,Zn SODand Trx-1 (ELISA) with impending labour and the predictive potential ofthese biomarkers was determined.Methods: Serial CVF samples were collected weekly from 73 healthy,multiparous women with singleton pregnancies beginning from 36 weeks’gestation until the onset of spontaneous labour (before membranerupture). Data was analysed after correction for total protein.Results: 193 samples were assayed for TAC (Fig 1A). TAC was 8-foldsignificantly lower in labour than >29 days prior to labour onset (ANOVA,p<0.001). TAC was about 2-fold significantly lower at 0-7, 8-14, 15-21 and22-28 days compared to >29 days before labour (ANOVA, p<0.001).Cu,Zn SOD was 1.5 to 1.9-fold significantly decreased in labour comparedto samples collected at 0-7, 8-14, 15-21 and 22-28 days before labour onset(Fig 1B, n¼170, ANOVA, p<0.001). Regression analysis indicated that Cu,ZnSOD significantly decreases with impending term labour (p¼0.003).Trx-1 was 2.8 to 5-fold significantly lower in labour compared to 0-7, 8-14,15-21 and 22-28 days before labour onset (n¼163, ANOVA, p¼0.002). Therewas a significant correlation between Cu,Zn SOD and Trx-1 concentrations(r¼0.433, p<0.001), and between Trx-1 and TAC (r¼0.349, p<0.001).Receiver Operator Characteristic (ROC) curves were produced for eachbiomarker and Binary Logistic Regression analysis was performed todetermine the efficacy of these biomarkers (singly and in combination) topredict labour within 3 days of onset (Table 1). The combination of TAC andCu,Zn SOD produced the best predictive efficacy with good sensitivity andvery high specificity. Positive and negative predictive values were superiorcompared to each biomarker alone.Discussion: These findings suggest that labour is associated withincreased oxidative stress even before its onset. These biomarkers couldserve as potential therapeutic targets as well as predictors of labour onset.Studies are in progress to determine the role of these biomarkers in thepreterm labour setting.

Table 1

TAC C
u,Zn SOD Trx-1 T AC andCu,Zn SOD
Cut off values
30 uM/mgtotal protein
0t

.5 ug/ml/mgotal protein

160 ng/ml/mgtotal protein

-

ROC - Areaunder curve

0.646 0
.687 0.646 0 .928
Sensitivity (%)
51.6 5 4.8 51.6 6 1.3 Specificity (%) 69.5 7 8.6 76.3 9 7.0 +ve Predictive
Value (%)
28.6 3 7.8 34.0 8 2.6
-ve PredictiveValue (%)

85.9 8
8.0 87.0 9 1.4
False +ve (%)
30.5 2 1.4 23.7 3 .1 False -ve (%) 48.4 4 5.2 48.4 3 8.7
Reference[1] Di Quinzio MKW et al, 2008, J Proteome Res, 7:1916-1921.Keywords: Labour, Oxidative Stress, Prediction, Cervico-vaginal fluid


[P14.08].INVOLVEMENT OF OXIDATIVE AND ANTI-OXIDATIVE STRESSES STRESSMECHANISMS IN PREGNANCY-INDUCED HYPERTENSION

Mitsuyo Matsumoto*, Osamu Akutagawa, Hirotaka Nishi, ChinatsuHiguma, Keiichi Isaka. Department of Obstetrics and Gynecology, TokyoMedical University, Japan

Objective: The body can clear reactive oxygen species and free radicalsgenerated under normal conditions through its antioxidative system. DNAdamage occurs when generation exceeds antioxidative capacity and isassociated with various disorders. It is becoming increasingly clear that thepathogenesis of pregnancy-induced hypertension (PIH) is associated withthe inflammatory response, oxidative stress, and anti-oxidative stress thatresults from reduced uteroplacental circulation. The purpose of this studywas to clarify the role of antioxidative stress in PIH pathogenesis.Methods: We explained the purpose of the study to participants, obtainedconsent, and examined amniotic fluid from the following tissues at variousstages: first trimester chorionic villi (18 cases), second trimester chorionicvilli (10 cases), normal placenta from Caesarean sections (12 cases), normalplacenta from vaginal births (10 cases), and placenta from PIH patients (15cases). We used Cu/Zn superoxide dismutase (SOD) and Mn-SOD as anti-oxidant indicators, and 8-OHdG as an oxidative stress indicator.Measurements were performed by ELISA, microplate assays, and real-timeRT-PCR.Results: SOD mRNA levels did not change by delivery method, but weresignificantly higher in first trimester chorionic villi compared to secondand third trimester chorionic villi. The PIH group had significantly lowerSOD mRNA and protein levels compared to the normal group, and the PIHgroup with intrauterine growth retardation showed an even more signif-icant decrease. In contrast, 8-OHdG levels were significantly higher in thePIH group compared to the normal group.Conclusion: Oxidative and anti-oxidative stresses are associated withvarious pathologies, including PIH. Our findings suggest the possibilitythat placental oxidative stress influences embryonic development throughan antioxidative mechanism.Keywords: pregnancy, pregnancy-induced hypertension, anti-oxidativestress, oxidative stress

[P14.09].MARKERS OF ENDOTHELIAL ACTIVATION IN HIGH RISK PREGNANCIES

Jana Prochazkova*1, Martin Prochazka2, Ludek Slavık2, Jana Ulehlova2,Ondrej Simetka3, Alena Mechurova4. 1Dept. of Hemato-oncology, MedicalFaculty of Palacky University, Czech Republic, 2Dept. of Obstet. andGynaecol, Medical Faculty of Palacky University, Czech Republic, 3Dept. ofObstet. and Gynaecol, Faculty Hospital Ostrava, Czech Republic, 4Institutefo the Care of Mother and Child, Prague, Czech Republic

Introduction: The hypertension and preeclampsia in pregnancy are mul-tisystemic diseases characterized by hypertension, proteinuria andgeneralized systemic vasoconstriction.The ischaemia of the fetoplacentalunit cause the release of specific factors into maternal vessels and subse-quent activation of the endothelium and vasoconstriction. There is a rushdevelopment of the laboratory tests and a new markers of the endothelialactivation have been found. Eg. t-PA, PAI-1, vWF, EPCR, thrombomodulinand endothelial microparticules with procoagulant activity.The aim of the study: To detect of above mentioned markers of endothelialactivation in healthy pregnant women compared to those with pregnancycomplicated by hypertension, diabetes mellitus and preeclampsia.The work hypothesis: We suppose that plasma specimens of the womenwith preeclampsia and diabetes mellitus will contatin a higher levels ofendothelial activation markers compared to healthy pregnant.Methods: All included patient have to assign an informed constent. Theblood samlping will be taken by the routine way at the time of the first bloodpregnancy sampling the end of the first trimester. The second specimen willbe taken between 24.- 28. weeks of gestation. The following tests will beperformed: t-PA – ELISA, PAI-1 – ELISA, vWF.Ag – EIA (immunologicdetection by imunoturbidimetry), ePCR – ELISA, MMP-2,9 – ELISA (fluo-rogenic detection), endothelial microparticules – Flow cytometry.Results: The levels of vWf Ag and vW activity are significantly different(P ¼ 0,02 resp. 0,01) in group patietns with diabetes in comparison withcontrol group.The levels of TRM are significantly different (P ¼ 0,03) in group patienswith hypertension as well as in group patietns with diabetes (P ¼ 0,02) incomparison with control group.The levels of ePCR are significantly different (P ¼ 0,04) in group patienswith hypertension as well as in group patietns with diabetes (P ¼ 0,01) incomparison with control group.The levels of tPA and PAI are not significantly different in group patienswith hypertension as well as in group patietns with diabetes in compar-ison with control group.Conclusion: As expected, there were stat. Significant differences in thediabetes group in the case of vWf Ag, vWact., thrombomodulin and ePCR.Significant diference was found in the hypertension group compared tocontrols in ePCR.Whereas EMP, MMP-9 were not different in all groups.Supported by the Grant of Min.of Health, Czech republikNR 9282-3/2007Keywords: Endothelium, Preeclampsia, Diabetes mellitus, Thrombosis


[P14.10].MATERNAL-FETAL DISTRIBUTION OF MINERALS IN TEENAGERSCOMPARED WITH ADULT WOMEN

MI Moraes, RF Barbosa, RE Santo, FS Santos, EFO Jesus, MG Tavares doCarmo*. Universidade Federal do Rio de Janeiro, Brazil

Introduction: Healthy intra-uterine development of the fetus requiresadequate amounts of minerals, which are only obtainable from maternalblood via placenta. Teenagers are still in growth and development andoften present inadequate nutrition habits, therefore their mineral intake isusually insufficient. The aim of this study was to compare calcium, iron,copper and zinc levels in maternal and cord plasma and in maternal andfetal placenta of teenagers and adult pregnant women.Methods: In Forty teenagers aged between 15 – 19 years and forty adultwomen, aged between 20 – 35 years samples were collected of maternalblood at day 2 postpartum and umbilical cord blood and maternal and fetalplacental tissues at birth. X-Ray Total Reflection Fluorescence Spectrom-etry was used for mineral analysis. The Mann-Whitney test was used tocompare plasma and placental concentrations of minerals.Results: Calcium, iron, copper, and zinc levels in maternal placenta pre-sented no difference between teenagers and adult women. However, theselevels in fetal placenta were higher in adult women than in teenagers. Inmaternal and cord plasma, calcium, copper and zinc levels presented nodifference between teenagers and adult women. Only iron levels werehigher in maternal and cord plasma of teenagers than in adult women.Discussion: Our results suggest that the distribution of minerals in thefetal placenta is different for teenagers and adult women. If there isa competition between mother and fetus in pregnancy in adolescencedeserves better elucidation in future research. We recommend studies onthe placental transport of these minerals.Keywords: minerals, teenagers, nutrition, maternal anda fetal placenta

[P14.11].POTENTIAL MAGNETIC RESONANCE IMAGING (MRI) BIOMARKERS OFPLACENTAL STRUCTURE

C Wright*1, DM Morris2, PN Baker1, IP Crocker1, GJ Parker2, CP Sibley1.1Maternal & Fetal Health Research Centre, University of Manchester,United Kingdom, 2Imaging Sciences & Biomedical Engineering, Universityof Manchester, United Kingdom, 3Magnetic Resonance Centre, Universityof Nottingham, United Kingdom

Placental insufficiency is a major cause of fetal growth restriction (FGR)and accumulating evidence indicates that several aspects of placentalstructure are altered in this condition (Sibley et al. Paed Res 2005;58:827).MRI provides quantitative indices that may be used in the non-invasiveassessment of the human placenta, such as relaxation time measurements,T1 and T2. In previous studies (at 0.5 T magnetic field strength) these havebeen found to be reduced in FGR (Gowland et al. Mag Res Imag1998;16:241). We hypothesised that these observed differences may relateto alterations in placental tissue structure and be more readily defined atincreased field strengths. Here, we report on the first phase of testing thishypothesis, in a study of women having normal pregnancy.Objectives: 1) To assess T1 and T2 relaxation time measurements andcorrelate these with morphometric analyses of the human placenta followingdelivery. 2) To assess placental heterogeneity by comparing relaxation timesand morphometric analyses in central and peripheral placental regions.Methods: 16 women with uncomplicated pregnancies underwent MRIexamination (1.5 T) between 20 and 40 weeks gestation. T1 and T2

measurements were acquired with whole placental coverage, co-localisedwith a structural scan. T1 and T2 values were calculated by combining 8points (3 central, 5 peripheral) across the placenta, central and peripheralregions were also analysed independently. Placental biopsies were taken at8 randomly sampled points (3 central, 5 peripheral). Formalin-fixed, wax-embedded sections were stained with hematoxylin and eosin and subjectedto image analysis. The areas occupied by villi and fibrin were quantified.Results: 7 of the 16 women (28-38 weeks at time of scan) had placentasavailable for analysis. A significant correlation was observed between villousarea and T2 (p¼0.048), but not T1 (p¼0.302) relaxation times. Correlationsbetween relaxation times and fibrin area or fibrin: villous area ratio were notsignificant, although trends for a fall in relaxation times with increasing fibrinwere seen. Placentas were heterogeneous, with no significant differencesbetween central and peripheral regions in villous or fibrin area, T1 or T2.

Conclusions: Villous area is known to be reduced in FGR (Daayana et al.J Soc Gynecol Investig. 2004;11(8):545). These early data suggest relaxa-tion times may be a useful biomarker of villous area and potentially usefulin identifying FGR in utero. We will continue to examine placentas fromnormal and FGR pregnancies, making comparisons with placentalheterogeneity, morphometry and function.Funded by the MRC.Keywords: MRI, IUGR, fetus, morphometry


[P14.12].A CASE OF GESTATIONAL TRANSIENT THYROTOXICOSIS IN TWINPREGNANCY COMPLICATED BY PANHYPOPITUITARISM

K. Takaishi*, M. Yamaguchi, K. Uchino, R. Honda, T. Ohba, H. Katabuchi.Kumamoto University, Japan

In a minority of pregnant women, the concentration of thyroxine increasesabove the upper normal value because of excess stimulation of the TSHreceptor by hCG. Here we report a case of gestational transient thyrotox-icosis in twin pregnancy complicated by panhypopituitarism. A 29-year-old woman was referred to our University Hospital with complaint ofinfertility. She had been treated for a craniopharyngioma at 25 years of age.Postsurgical hormone replacement was carried for panhypopituitarismwith diabetes insipidus. A diagnosis of pituitary amenorrhea was madeafter gynecological evaluations, and the patient underwent treatment forinfertility. The patient became pregnant with dichorionic diamniotic twinsduring hMG-hCG treatment cycle with AIH. The patient was complicatedby frequent vomiting and weight loss after 5 weeks of gestation and thepatient was admitted to our hospital. A diagnosis of hyperemesis grav-idarum was made at 8 weeks of gestation. Laboratory tests showedelevated free T3 and T4, and with absent TSH and thyroid autoantibodies.Adrenal failure was excluded because hydrocortisone administration wasineffective. A diagnosis of gestational transient thyrotoxicosis was made.The patient’s symptoms improved after the discontinuation of thyroxinesupplementation and the decrease in serum hCG. The patient had a vaginaltwin delivery at 38 weeks of gestation, and thyroxine supplementationwas resumed on the third postpartum day. The patient’s serum hCGstrongly correlated with the serum free T4 under conditions of low TSHsecretion. In conclusion, gestational thyrotoxicosis was triggered despitethe lack of endogenous TSH. This case proves that TSH receptor is capableof being stimulated by endogenous hCG.Keywords: gestational transient thyrotoxicosis, human chorionic gonad-otropin, thyroid stimulating hormone receptor

[P14.13].PATERNAL RENIN ANGIOTENSIN SYSTEM POLYMORPHISMS AREASSOCIATED WITH PREGNANCY COMPLICATIONS

A Zhou*, S Thompson, R Nowak, J Zhang, G.A. Dekker, C.T. Roberts. ResearchCentre for Reproductive Health, Discipline of Obstetrics and Gynaecology,University of Adelaide, Australia

Introduction: Preeclampsia (PE), small for gestational age (SGA) and pre-term birth (PTB) together affect 20% of first pregnancies. Currently there isno reliable way to identify women at risk. Polymorphisms in genes in therenin angiotensin system (RAS) may be associated with impaired placen-tation and poor maternal response to pregnancy and hence predict risk forpregnancy complications. We aimed to determine if three functionalpolymorphisms in RAS genes, namely, AGT T174M, ACE A11860G and AT1RA1166C are associated with pregnancy complications.Methods: Pregnancy trios were prospectively recruited from Lyell McEwinHealth Service in Adelaide. Pregnancies were classified into normal(n¼258), PE (n¼38), SGA (n¼60), gestational hypertension (GH, n¼51),PTB (n¼32) and gestational diabetes (GD, n¼13). Parental blood andmaternal blood pressure were sampled or measured at 15 weeks gestation.Cord blood was sampled after delivery. DNA was extracted from buffy coatsand genotyped by the Australian Genome Research Facility utilizing theSequenom MassARRAY system. Maternal plasma [ACE] was measured byELISA. Data were analysed by ANOVA, Chi-square and Fisher’s exact test.Likelihood ratios (LR) were calculated.Results: None of the RAS maternal and neonatal genotypes were asso-ciated with pregnancy complications. For maternal ACE A11860G, plasma[ACE] in women with GG was 24% and 74% higher than AG (p¼0.03) andAA (p¼0.001), respectively. A positive correlation between plasma [ACE]and mean arterial pressure (r¼0.378, p¼0.003) was observed in normalpregnancy. However, maternal ACE genotype did not correlate withblood pressure at 15 weeks gestation. Of great interest, paternal AGTT174M, AT1R A1166C and ACE A11860G were associated with GD(p¼0.028, LR¼5.0), PTB (p¼0.032, LR¼7.3) and PE (p¼0.038, LR¼6.7),respectively.Discussion: Our data suggest that paternal genotype for genes in the reninangiotensin system may be important in determining risk for pregnancycomplications, consistent with the role of paternity in their aetiology.Keywords: Placenta, Pregnancy complications, Renin angiotensin system,Paternal genotype


[P14.14].THE EFFECTS OF FETAL HYPERINSULINAEMIA ON VEGF-165 ANDVASCULAR PERMEABILITY IN THE PERFUSED HUMAN PLACENTA

J Lucas, SP Walker, PEB Rodgers, F Sciota, L Leach*. University of Notting-ham, United Kingdom

Insulin is known to upregulate vascular endothelial growth factor (VEGF-A), a potent enhancer of vascular permeability. In diabetic pregnancies,fetal hyperinsulinaemia may be one of the factors leading to elevatedlevels of VEGF observed in the placenta, with its resultant leaky vascularphenotype. The aim of this study was to assess levels of VEGF-165a and itssplice variant VEGF 165b and the integrity of junctions in fetal vessels aftera brief hyperinsulinaemic insult.Using an established method, term human placenta from normal preg-nancies (obtained by elective Caesarean section) were perfused for 20minutes with (n¼3) or without (n¼3) 25mU/mL insulin in the fetal circu-lation. In the last 10 min, TRITC-dextran (76Mr) was added to the fetalcirculation in both groups. After perfusion fixation, tissue biopsies weresubjected to immunocytochemistry to localise VEGF isoforms, junctionalb-catenin and occludin and tracer ‘‘hot spots’’. Selected random samplingwas used to count the % of vessels in terminal, intermediate or stem villidisplaying positive immunoreactivity.In the insulin group, analysis of large vessels in stem villi and microvesselsin intermediate and terminal villi revealed increases in percentage of allVEGF-positive vascular profiles (p<0.05), whilst there were decreases inVEGF165b-positive large vessels. Increased vascular leakage was seen inboth large and microvessels in the insulin group. This was accompaniedwith a downregulation of b-catenin in all vessels and downregulation ofthe tight junctional molecule, occudin in large vessels. These combineddata suggest that brief fetal hyperinsulinaemia is capable of affecting bothadherens and tight junctions, altering the phenotype of chorionic vesselsthroughout the villous tree. Beyond implications in placental barrierfunction, the results raise the possibility that chronic high fetal insulin mayalter the fetal vascular phenotype, with implied potentially long-lastingeffects on the infant.Funded by ASGBIKeywords: fetal insulin, VEGF, tracer leakage, feto-placental vessels

[P15.01].SCANNING ELECTRONE MICROSCOPY IN TIME OF PROPOSEDIMPLANTATION BY ENDOMETRIAL PATHOLOGY

O. ILINA*, T. ZADOROGHNA, I. SUDOMA, I. ILYIN. 1Institute of Paediatrics,Obstetrics and Gynecology, Ukraine, 2Clinic "Nadiya", Ukraine, 3Institute ofGenetic Reproduction, Ukraine

Aim of the study: to investigate peculiarities of cilliar cells and pinopodesformation in endometrial luminal epithelia in women with pathologicalstatements of endometrium.Object: double Pippelle – biopsies of endometrium from 360 women intime of implantation window with different types of endometrialpathology: hypotrophy, hyperplasia, chronic endometritis, asynchrony ofendometrial maturation.Methods: Scanning electrone microscopy (JEOL Superprobe 733) androutine histological investigation of endometrial samples of every womanon two terms.Results: Defects of pinopodes (uterodomes) formation and developmentwas more significant by endometrial hypotrophy. Pathological changes ofcilliar cells in quantity and structure were observed by endometrialhyperplasia.Summary: Significant changes in luminal epithelia in time of implantationwindow in women with endometrial pathology were revealed by scanningelectrone microscopy that may cause infertility in these women andshould be taken into consideration by diagnostic and treatment.Keywords: Scanning electron microscopy, implantation, endometrialpathology, infertility


[P15.02].CHANGES IN VEGF-C, VEGF-D, VEGF-R2 AND VEGF-R3 IN NON-PREGNANT HUMAN ENDOMETRIUM DURING THE MENSTRUAL CYCLEAND IN WOMEN WITH RECURRENT MISCARRIAGE

GE Lash*1, RK Adcock1, BA Innes1, JA Drury2, S Quenby2, JN Bulmer1.1Newcastle University, United Kingdom, 2University of Liverpool, UnitedKingdom

Introduction: Endometrial vascular development is a critical process.Spiral arteriole vascular smooth muscle cell (VSMC) numbers and differ-entiation state increase throughout the menstrual cycle. In some womenwith recurrent miscarriage (RM) vascular development is enhanced. VEGF-C and -D have both angiogenic (via VEGF-R2) and lymphangiogenic (viaVEGF-R3) properties.Aim: Determine the temporal and spatial expression of VEGF-C, -D, -R2,and -R3 in endometrium throughout the menstrual cycle and in womenwith RM.Methods: Endometrial biopsies taken at the time of hysterectomy (n¼7proliferative (P), n¼4 early secretory (ES), n¼7 mid-late secretory (LS)) andendometrial pipelle biopsies from women with RM at LH+7 (n¼14 LS) wereimmunostained for VEGF-C, -D, -R2, and -R3 and assessed using quick-score. Stroma, VSMC, endothelial cells (EC) and glandular epithelium (GE)were scored separately. Scores for all cell types were added together togive a ‘total’ score.Results: Endometrium immunostained positive for all factors. VEGF-Cimmunostaining was reduced in LS compared to P (total P¼0.009; ECP¼0.0009; GE P¼0.001) and ES (total P¼0.02; EC P¼0.005). No changes inVEGF-D expression were observed, and VEGF-D was not detected in VSMC.VSMC immunostaining for VEGF-R2 was increased in ES (P¼0.003) and LS(P¼0.01) compared to P. VEGF-R2 was decreased in RM compared to LS(total P¼0.003; stroma P¼0.009; VSMC P¼0.0003). Stromal immunos-taining for VEGF-R3 was reduced in ES (P¼0.03) and LS (P¼0.03) comparedto P. GE immunostaining for VEGF-R3 was decreased in LS compared to P(P¼0.004), but was increased in RM compared to LS (P¼0.02).Discussion: VEGF-C, -R2 and -R3 are temporally and spatially regulatedduring the menstrual cycle. These factors are likely involved in endome-trial regeneration. VEGF-R2 and VEGF-R3 expression was widespread inendometrium suggesting importance of their ligands in endometrialfunction. The significance of reduced VEGF-R2 expression in RM needs tobe further explored.Keywords: angiogenic growth factors, non-pregnant endometrium,recurrent miscarriage

[P16.01].PLACENTAL TROPHOBLAST DYNAMICS IN SUDDEN INFANT DEATHSYNDROME (SIDS)

T Ansari*1, B O’Neill2, JE Gillan2. 1Dept. of Surgical Research, NPIMR,Harrow, London, United Kingdom, 2Dept. of Histopathology, RotundaHospital, Dublin, Ireland

Introduction: SIDS occurs postnatally but the origins are hypothesised tooccur in utero resulting from delayed or arrested developmental homeo-stasis. The degree of maternal–foetal exchange in utero is paralleled by anincreased developmental complexity of the placenta. Specifically, duringthe third trimester placental villous growth experiences an exponentialincrease in transfer capacity to match the growing demands of the foetus.It is during this period that perturbations in villous trophoblast kinetics arelikely to have the greatest impact on foetal organogenesis.Methods: SIDS placentae were retrieved from archived storage and sub-divided into two groups based on a birth weight above (SIDS NBW n¼12)or below (SIDS LBW n¼12 the 10th centile for gestational age andcompared with term controls (n¼12). Uniform random sampling wasemployed and tissue samples used to produce 5mm H&E stained sections.Stereologically derived volumes of the following features were estimated:cytotrophoblast (CT), syncytiotrophoblast (ST) and syncytial knots (SK)-both apoptotic and non-apoptotic for all villous types.Results: Total trophoblast volume was increased (P¼0.007) in SIDS NBWplacenta, originating from significant increases in both CT (P¼0.004) stemand terminal villi) and ST (P¼0.031; stem villi) volumes. In contrast, SIDSLBW placentae showed no significant change in total trophoblast volumeor its subcomponents; however, CT volume showed a trend towards anincrease (stem villi). An increase in total apoptotic SK volume wasobserved in SIDS NBW (intermediate and terminal villi) and SIDS LBW(intermediate villi). Histologically, the CT nuclear size appeared larger andexisted in places as a multi-layered arrangement.Discussion: Although the trophoblast layer is thicker in SIDS NBW theincreased volume of focal apoptotic SK suggests increase extrusion tomaintain a minimum diffusion distance for oxygen and nutrient transfer.Trophoblast kinetics in SIDS NBW and SIDS LBW differ with the formerinitiating an adaptive mechanism to maintain foetal growth.Keywords: SIDS


[P16.02].CYTOKERATIN AND CEA EXPRESSION IN VILLOUS CHORION

T. Archakova*, T. Zadoroghna. Institute of Paediatrics, Obstetrics andGynecology, Ukraine

Aim of the study was to investigate Cytokeratin and CEA expressionpeculiarities in villous chorion by physiological pregnancy (II trimester).Object: 20 biopsies from healthy women with physiological pregnancy,who had a chromosomal pathology were observed.Methods: immunohistochemical investigation with monoclonal anti-bodies to Cytokeratin Clones AE1/AE3 and CEA (DAKO) on paraffin –embded cuts.Results: Cytokeratin expression was high in cytotrophoblast, vascularendothelium, stromal cells and trophoblastic proliferates; moderate – insincitiotrophoblast. CEA expression was insignificant and was revealed asweak or at times - moderate in sincitium and cytotrophoblast of isolatedintermediate differentiated villi.Summary: Strongly marked Cytokeratin expression and weak expressionof CEA were observed in II trimester placenta, that characterize one ofplacental histogenesis way.Keywords: Cytokeratin, CEA, chorion, placental histogenesis

[P16.04].CORD BLOOD CYTOKINE AND CHEMOKINE PROFILE IN PREGNANCIESCOMPLICATED BY ASTHMA

A Osei-Kumah*, N Hodyl, V Clifton. University of Adelaide, RobinsonInstitute, Australia

Introduction: Fetal exposure to an adverse intrauterine environment canincrease susceptibility towards disease in adult life. Furthermore maternalasthma is an increased risk factor for the development of atopy or asthmain the offspring. There is evidence that this is associated with differentialplacental gene expression. We have demonstrated that maternal asthma isassociated with increased basal cytokine expression in the placentae offemale neonates compared to controls. Such placental inflammatoryresponses may program the development of the fetal immune system. Theaim of current study was to assess the cytokine and chemokine profile incord blood from pregnancies complicated by maternal asthma.Methods: Following delivery, cord blood was collected from controlpregnancies (n¼7), pregnancies complicated by untreated asthma (n¼14)and those treated with inhaled glucocorticoids (n¼17). Plasma wasassayed for 19 cytokines and chemokines with a Lincoplex assay kit ona Luminex multi-measurement platform.Results: Asthma was associated with an increased cord blood concentra-tion of granulocyte colony stimulating factor (G-CSF) compared to controls(p¼0.003). In cord blood from females, increased concentrations of inter-leukin (IL)-10 (p¼0.038) and IL-12 (p¼0.025) were also demonstrated. Incord blood from males, cytokine and chemokine concentrations did notchange with asthma. None of the measured cytokines or chemokines wereaffected by inhaled glucocorticoids for asthma treatment.Conclusion: Increased cytokine concentrations were observed in cordblood from females following maternal asthma. This data suggests thatthe developing immune system may be programmed differentially infemales and males, given that G-CSF stimulates the production of hae-matopoietic stem cells, and both IL-10 and IL-12 are involved in celldifferentiation. These findings may help elucidate mechanisms contrib-uting to increased rates of allergy and atopy currently observed in femalechildren.Keywords: cord blood, cytokine, allergy, programming

[P16.05].IS MATERNAL PROGESTERONE ACTUALLY INDEPENDENT OF THE FETALSTEROIDS?

Antonin Parizek*, Andrea Paskova, Martin Hill, Michaela Klimkova, MartaKalousova, Luboslav Starka. Department of Obstetrics and Gynecology ofthe First Faculty of Medicine and General Teaching Hospital, Prague, CzechRepublic

Progesterone and estradiol are foremost steroid hormones in humanpregnancy; however, the origin of maternal progesterone has not been stillsatisfactorily explained despite the generally accepted opinion thatmaternal LDL-cholesterol is a single substrate for placental synthesis ofmaternal progesterone. There is still a question: ‘‘Why the levels ofprogesterone are substantially higher in fetal and not in maternal blood?’’Hence, the role of the fetal zone of the fetal adrenal (FZFA) in the synthesisof progesterone precursors was addressed.The FZFA may be directly regulated by placental CRH inducing an excessiveproduction of sulfated 3b-hydroxy-5-ene steroids like sulfates of dehy-droepiandrosterone (DHEAS) and pregnenolone (PregS).To our hypothesis, besides the necessity to synthesize de novo all thematernal progesterone from cholesterol, it may be more convenient toutilize the fetal PregS. The activities of sulfatase and 3b-HSD aresubstantially higher than those ones of cytochrome P450scc and are notrate-limiting for the placental progesterone synthesis.Keywords: steroids, labor, progesterone, metabolome


[P16.06].EFFECTS OF MATERNAL MOOD AND ANTIDEPRESSANT USE ONPLACENTAL GENE EXPRESSION

KL Ponder*1,2, AL Salisbury1,2, BG McGonnigal1,2, AM LaLiberte2, JF Pad-bury1,2. 1Warren Alpert Medical School of Brown University, United States,2Department of Pediatrics, Women & Infants’ Hospital, United States

Introduction: Previous work has shown that stress, drug exposure andgrowth restriction modify placental neuroendocrine gene expression. It isnot known whether maternal mood disorders (depression/anxiety) andantidepressant treatment have similar effects and influence fetalprogramming. We sought to determine how maternal depression, anxiety,and treatment with Selective Serotonin Reuptake Inhibitors (SSRIs) affectplacental human serotonin transporter (hSERT), norepinephrine trans-porter (hNET), and 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD-2) expression.Methods: We collected placental samples from 52 controls, 56 womenwith depression and/or anxiety symptoms during pregnancy, and 27women who used SSRIs in at least the third trimester. A subset of 25women who consented to structured interview was also included. Inven-tory of Depressive Symptomatology and Hamilton anxiety scores weredetermined. RNA expression was determined by real-time PCR. ANOVAwas used to determine statistical significance between control, depres-sion/anxiety, and SSRI groups.Results: Exclusions included other psychotropic medications, nicotine orillicit drug use, diabetes, and gestational age <37 weeks. There wasa significant overall effect of mood and SSRIs on expression of SERT, NET,and 11beta-HSD-2. 11beta-HSD-2 expression was significantly increased inthe depression/anxiety group, but not the SSRI group, compared tocontrols (p<.05). NET was positively correlated with 11beta-HSD-2 andSERT expression (p<0.001).Discussion: Maternal mood has a significant effect on placental 11beta-HSD-2 gene expression. 11beta-HSD-2 is induced by cortisol and convertscortisol to its inactive form. Placental 11beta-HSD-2 therefore serves asa barrier to maternal cortisol passage to the fetus. Depression is associatedwith increased blood cortisol levels. The increase in 11beta-HSD-2expression in the depression/anxiety group could represent a compensa-tory response to increased maternal cortisol levels. This finding isconsistent with previous in vitro studies in human trophoblast cells. Theseobservations suggest possible mechanisms for long term effects ofmaternal mood and antidepressant treatment on fetal/neonatal neuro-developmental programming.Keywords: gene expression, depression, antidepressants, fetalprogramming

[P16.07].ADVANCED PATERNAL AGE AFFECTS BIRTH WEIGHT AND PLACENTALTHICKNESS OF FEMALE BUT NOT MALE INFANTS: AN ANALYSIS OFTHE COLLABORATIVE PERINATAL PROJECT (CPP)

CM Salafia*1,2, DP Misra3, AK Charles4, RK Miller5. 1Placental Analytics LLC,United States, 2Institute for Basic Research, United States, 3Wayne StateUniversity, United States, 4Princess Margaret Hospital, Australia, 5Univer-sity of Rochester, United States

Background: Advanced paternal age has recently been identified as a riskfactor in female offspring for neurodevelopmental and neuropsychiatricoutcomes such as autism and schizophrenia. The mechanism has beenspeculated to involve age-pathology of the paternally-derived X chro-mosome. We tested the hypothesis that advanced paternal age wouldaffect the placenta (in which paternal genes are preferentially expressed)of female but not male infants, and that those placentas would yielda smaller birthweight than placentas from pregnancies with a youngerfather.Methods: 11,120 male and 10,542 female newborns had complete data forchorionic larger and smaller diameters, disk thickness, distance from cordinsertion to the disk edge, cord length, placental weight, and categoriza-tion of placental shape as ‘‘round/oval’’ versus ‘‘other’’, and parental ages.Univariate and multivariate linear regressions were performed.Results: In bivariate regression, advanced paternal age was associatedwith greater birth weight in males (p<0.001) and females (p<0.0001). Inmale infants, paternal age effects disappeared after adjustment formaternal age, while in female infants, such adjustment led to a significantnegative effect of paternal age (p<0.0001). Paternal age also showed aninverse relationship to disk thickness in female (p<0.0001) but not in maleinfants (p¼0.62). Paternal age was not associated with placental weight orchorionic disk area (calculated from the larger and smaller placentaldiameters) in either sex.Conclusion: Advanced paternal age is a risk factor for autism andschizophrenia, in females. Autism and schizophrenia are also disorders ofabnormal neuronal connectivity. One speculation has been that thepaternally derived X chromosome carries altered/mutated genes and/orabnormal/altered epigenetic programming. We propose that the observedeffect of thinner (less well arborized) placentas of girlswith older fathersmirror fetal branching in other organs, including neurons. Study of theplacenta may therefore be a proxy for analysis of the brain proper.Keywords: paternal age, placental growth, birth weight, fetal programming


[P16.09].BIRTHWEIGHTS SMALLER OR LARGER THAN THE PLACENTA WOULD‘‘PREDICT’’ IS ASSOCIATED WITH DIASTOLIC BLOOD PRESSURE AT AGE7 YEARS: AN ANALYSIS OF THE COLLABORATIVE PERINATAL PROJECT(CPP)

CM Salafia*1,2, DP Misra3, AK Charles4, RK Miller5. 1Placental Analytics LLC,United States, 2Institute for Basic Research, United States, 3Wayne StateUniversity, United States, 4Princess Margaret Hospital, Australia, 5Univer-sity of Rochester, United States

Background: Fetal growth is a complex process depending on parentalgenetics, maternal /uterine environment and placental function. Placentalgrowth measures collected in the CPP (disk shape, larger and smallerdiameters, disk thickness, site of cord insertion and cord length) accountfor w40% of birthweight variance and have been shown to have inde-pendent effects on birth weight. (Salafia CM et al. Birth Defects Res A ClinMol Teratol. 2007;79(4):281-8.) We hypothesized that altered placentalproportions (and different vascular structure) affect the fetal cardiovas-cular system, and be reflected in variation in childhood blood pressure.Methods: Using linear regression, we generated a birth weight pre-dicted by the available placental measures (disk shape, smaller andlarger diameters, disk thickness, site of cord insertion, and cord length).The ratio of the actual birth weight to that predicted by placentalparameters (observed/expected ratio, OER) was used as the independentvariable in analyses of age 7 year diastolic and systolic blood pressures inthe 15,902 singleton liveborns delivered between 34-42 weeks withcomplete.Results: After adjustment for precise ages at blood pressure measurement,the OER had a significant independent effect on diastolic blood pressure(b¼3.4þ.63, p<0.001) at 7 years of age. This effect persisted after furtheradjustment for gestational age, sex, socioeconomic status, and maternalpre-pregnancy height and weight, and was also independent of BMI,previously shown to be correlated with OER.Conclusion: Placental proportions, proxied here by the observed/expectedratio, reflect the vascular architecture of the arborizing placenta; differentproportions imply different numbers and distributions of vessels atdifferent levels of the villous tree. We interpret our findings as consistentwith the hypothesis that placentas with different vascular compositionaffect fetal cardiac work and/or reflect differences in intrinsic patterns ofthe cardiovascular system, and may either mediate or mark differences inchildhood diastolic blood pressure.Keywords: blood pressure, placental growth, birth weight, fetalprogramming

[P17.01].PRENATAL ETHANOL EXPOSURE IN THE RAT RESULTS IN GROWTHRESTRICTION AND DECREASED AQUAPORIN-1 GENE EXPRESSION INFEMALE BUT NOT MALE PLACENTAE

LB Wilson, DG Simmons, ME Probyn, KM Moritz*. University of Queens-land, Australia

Background: Exposure of the developing foetus to alcohol (ethanol) canresult in fetal growth restriction and impaired development. This mayoccur in part due to effects of ethanol directly on the placenta. High dosesof ethanol during pregnancy in animal models can reduce placental weightand function. In this study the effects of maternal consumption of a low tomoderate amount of ethanol throughout pregnancy on placental devel-opment was examined in a rat model.Methods: Pregnant rats were fed a control diet or a diet containing 6% (vol/vol) ethanol (ETOH). At day 20 of pregnancy, dams were killed and foetusesand placentae collected and weighed. RNA was extracted from the laby-rinth and gene expression of Glut-1, aquaporin-1 (AQP-1) and alcoholdehydrogenase (ADH) examined using real-time PCR. Placentae from maleand female foetuses were examined separately.Results: ETOH exposed females, but not males, were lighter than controlfoetuses (P<0.05). The placentae from all offspring (male, female, control,ethanol) were of a similar weight. ETOH resulted in decreased Aqp1ex-pression in the placenta of female but not male foetuses and tended todecrease ADH expression in both sexes. Gene expression is shown in thetable below (*P<0.05):

Gene Control (n¼7-10 samples Ethanol (n¼7-10 samples
from 5 litters) from 4 litters)
Glut1
Male 1.02�0.19 1.18�0.2 Female 1.24�0.18 1.25�0.18
Aqp1
Male 1.08�0.23 0.68�0.25 Female 1.36�0.23 0.60�0.21*
ADH
Male 1.13�0.24 0.75�0.43 Female 1.00�0.19 0.43�0.16
Discussion: Prenatal ethanol exposure has caused sex specific decreases inplacental Aqp1 gene expression and fetal growth. Aqp1 is expressed invascular endothelium and thus decreased expression may reflectdecreased development of the vasculature in the placenta of femalefoetuses thereby contributing to the observed growth restriction.Keywords: alcohol, aquaporin 1, sex


[P17.02].SEX SPECIFIC DIFFERENCES IN HUMAN PLACENTAL MICRO RNAEXPRESSION

A Osei-Kumah*, N Hodyl, W Kong, J Owens, V Clifton. University ofAdelaide, Robinson Institute, Australia

Introduction: We have previously identified sex specific differences inplacental gene expression associated with strategies for optimal growth andfetal survival. Specifically, we have shown differences in placental cytokinemRNA and glucocorticoid receptor expression. We have also identified sexspecific differences in global placental gene expression. Micro RNAs (miRs)are non-coding small RNAs and act as important post-transcriptionalregulators of gene expression by altering the abundance or translationalefficiency of mRNAs. The current study aims to examine miR expression inthe placenta, to determine if they are involved in the regulation of differ-ential gene expression observed in male and female placentae.Hypothesis: We hypothesise that post-transcriptional regulation of genesby miRs play an important role in conferring sexual dimorphism inplacental gene expression during development.Methods: RNA was extracted from male (n¼6) and female (n¼6) placentafrom normal pregnancies and miR analysis was conducted using exiqonarrays. Target prediction algorithm database was used to identify predictedtargets for identified miRs. Ingenuity Pathways Analysis (IPA) software wasused to identify functional networks of differentially expressed miRs.Results: One hundred and six miRs were differentially expressed betweenmale and female placentae based on a P-value of less than 0.05. Sixty miRswere up-regulated and 46 were down-regulated in female placentaecompared with males. Most of the differentially expressed miRS were clus-tered on chromosomes 13,14,16,17,19, 20 and X. Pathway analysis identifiednetworks involved in innate immune activation, cytokine signalling, gluco-corticoid receptor signalling, cellular growth and proliferation.Conclusions: There are differentially expressed miRs in male and femaleplacenta which target genes involved in cytokine gene expression andother immune pathways in the placenta. This may be related to thedifferential gene regulatory mechanisms initiated by males and femalesin-utero for growth and survival.Keywords: micro RNA, placenta, gene expression, cytokine

[P17.03].EFFECT OF TESTOSTERONE ON PLACENTAL CYTOKINE PRODUCTIONAND p38MAPKINASE

M Stark*1, N Hodyl1, N Scott2, E Green2, A Osei-Kumah1, V Clifton1. 1TheUniversity of Adelaide, Australia, 2The University of Newcastle, Australia

Introduction: Testosterone is known to have a suppressive effect oninnate immune function that may mediate the differential responses wehave previously demonstrated in pro-inflammatory cytokine expressionbetween male and female placenta. Activation of p38MAPkinase is centralto pro-inflammatory cytokine production. We questioned whetherplacental cytokine production and p38MAPkinase activity differed ina sex-specific manner, and were altered in the presence of testosterone.Hypotheses: We hypothesise that placental TNFa production will begreater in females than males due to increased activation of the p38MAPkinase signalling pathway. This sexually dimorphic production ofTNFa will be decreased in female placentae in the presence of testosterone.Methods: Placentae were collected from women pregnant with a male(n¼9) or female (n¼7) infant. Placental p38MAP kinase protein andactivity were measured by Western blot and activity assay, respectively.Placental explants were stimulated with LPS (1ng/ml) in the presence andabsence of testosterone. Supernatant was collected and tumour necrosisfactor (TNF) alpha concentrations were determined (ELISA).Results: Placental p38MAPkinase levels and activity were increased infemales compared to males (p<0.05). Following LPS stimulation, increasedTNFa concentrations were observed in placentae from females comparedto males (p<0.05). This was significantly reduced by co-treatment ofexplants with testosterone (p<0.05), to a level equivalent to males.Conclusions: This data suggests that the sex-specific levels and activationof p38MAPkinase results in increased TNFa production in femalecompared to male placentae. Testosterone appears to play a key regulatoryrole in this pathway, as the sex-specific differences in cytokine productionare abolished in its presence. This may be due to testosterone mediatedincreases in p38 MAP kinase phosphorylation that has been previouslyreported in the adult human literature. Whether a similar mechanismexists in the human placenta is a focus of our ongoing research.Keywords: cytokine, testosterone, p38MAP kinase, sex differences


[P18.01].IL-6 PROMOTES SHEDDING OF NECROTIC TROPHOBLASTS FROMPLACENTAL EXPLANTS

LM Chen*1,2, B Liu2, HB Zhao1, P Stone2, L Chamley2, Q Chen1,2. 1TheHospital of Obstetrics & Gynaecology, Fudan University, China, 2Depart-ment of Obstetrics & Gynaecology, The University of Auckland, NewZealand

Preeclampsia (PE) is characterised by elevated maternal blood pressure,preceded by endothelial cell dysfunction. Trophoblasts shed from theplacenta may be one of the factors that trigger PE. Women with PEfrequently have elevated serum levels of inflammatory markers such as, IL-6 and TNF alpha but their functional significance is unclear. In this studywe investigated whether these or other cytokines can alter trophoblastshedding.Placental explants were treated with 9 different cytokines for 72 hours.Shed trophoblasts then were harvested using our published method. Thenumbers of trophoblasts shed were quantified by automated cell counter.Expression of active of caspases 3&7 by the shed trophoblasts was deter-mined using a FLICA kit and confocal microscopy. The trophoblasts shedfrom cytokine-treated or control explants were exposed to endothelial cellmonolayers and endothelial activation determined ELISA for cell surfaceICAM-1.Treatment of explants with IL-6 caused a 50% increase (p¼0.001), whileTNF alpha and TGFbeta1, caused smaller but statistically significantincreases in the numbers of trophoblasts shed from explants. Trophoblastsshed from explants treated with IL-6, TGF beta1, or TGFbeta3 expressedsignificantly less active caspases 3&7 than controls or trophoblasts shedfrom explants treated with other cytokines. Exposing trophoblasts shedfrom IL-6- or TGFbeta1-treated explants to endothelial cells causeda significant (P<0.001) increase in endothelial cell-surface ICAM-1compared to controls.Normally trophoblasts shed from the placenta die by an apoptosis-likeprocess and their phagocytosis is silent but a shift to shedding of necrotictrophoblasts can lead to endothelial cell activation. It remains unclearwhat might trigger a shift from apoptotic to necrotic trophoblast death.This study suggests that IL-6 and possibly other cytokines can alter boththe number and the nature of shed trophoblasts such that, the tropho-blasts are more necrotic and their phagocytosis by maternal endothelialcells could contribute to the pathogenesis of preeclampsia.Keywords: cytokines, trophoblast deportation, endothelium

[P18.02].MEGALIN, A LDL RECEPTOR, MEDIATES INTERNALISATION OFANTIPHOSPHOLIPID ANTIBODIES INTO SYNCYTIOTROPHOBLAST

Q Chen*1,2, B Liu1, LM Chen2, P Stone1, LW Chamley1. 1Department ofObstetrics & Gynaecology, The University of Auckland, New Zealand, 2TheHospital of Obstetrics & Gynaecology, Fudan University, China

Antiphospholipid autoantibodies (aPL) increase the risk of a womendeveloping preeclampsia nine fold. Previously we have shown thatadding aPL to placental explants resulted in the internalisation of the aPLinto the syncytiotrophoblast and subsequently shed syncytial knots. TheaPL also increased the number of necrotic syncytial knots shed from theexplants. These necrotic syncytial knots in turn activated endothelialcells. The mechanism by which aPL were internalised into the syncy-tiotrophoblasts is unclear but aPL bind to the plasma protein b2 glyco-protein I which is transported into epithelial cells via megalin, anendocytic LDL receptor. Here we investigated whether aPL are internal-ised into the syncytiotrophoblast via megalin. Placental explants weretreated with monoclonal aPL (ID2 or IIC5) or a control antibody (CD45) inthe presence or absence of the megalin inhibitors, receptor associatedprotein (RAP), or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB).Internalization of the antibodies was monitored by confocal microscopy.Effects on endothelial cell activation were determined by addingtrophoblasts shed from explants treated with aPL, or a control antibody,+/- megalin inhibitors, to endothelial cell monolayers and monitoring theexpression of cell surface ICAM-1 by ELISA. Confocal microscopyconfirmed that aPL, but not control antibodies, were internalised into thetrophoblasts and that both megalin inhibitors blocked this internal-isation. Furthermore, trophoblasts shed from explants treated with aPL,but not control antibodies, activated endothelial cell monolayers whiletrophoblasts shed from explants treated with aPL plus megalin inhibitorsdid not activate endothelial cells.These results suggest that megalin is involved in the selective transport ofaPL in the syncytiotrophoblast and that once internalised, via megalin, aPLlead to aberrant shedding of trophoblasts. Since both altered trophoblastshedding and endothelial cell activation are thought to be involved in thepathogenesis of preeclampsia this might explain why aPL predisposewomen to developing preeclampsia.Keywords: antiphospholipid antibodies, megalin, internalisation, endo-thelial cell activation


[P18.03].THE ABILITY OF TROPHOBLASTS TO INTERNALIZE ANTIPHOSPHOLIPIDANTIBODIES CORRELATES WITH MEGALIN EXPRESSION

C Viall*, Q Chen, S Lau, P Stone, L Chamley. Department of Obstetrics &Gynaecology, The University of Auckland, New Zealand

Antiphospholipid antibodies (aPL) are autoantibodies that, by an unknownmechanism, increase the risk of developing preeclampsia nine fold. Wehave recently shown that aPL cause an increase in the number of syncytialknots shed from placental explants and a change in the trophoblast deathprocess towards a more necrosis-like death, which might contribute to thepathogenesis of preeclampsia. Prior to inducing changes in trophoblastsaPL, but not control antibodies, are internalised into the syncytiotropho-blast of explants suggesting a specific mechanism for internalisation of theaPL. Internalisation of aPL into the syncytiotrophoblast appears to berequired for aPL to affect trophoblast shedding and we have preliminaryevidence that aPL-internalisation is mediated by the endocytic receptor,megalin.Monoclonal aPL IIC5 (13mg/ml)or ID2 (6mg/ml), were incubated withmonolayers of the choriocarcinoma cell lines, Jar, Jeg-3, BeWo, or firsttrimester placental explants for 24 hours. Internalisation of aPL intotrophoblasts was visualised following fluorescent immuno-staining. Theexpression of megalin by the cell lines and explants was examined byimmuno-staining using an affinity-purified rabbit anti-megalin (Sigma).Experiments were repeated at least three times.Confocal microscopy demonstrated that BeWo cells, but not Jar or Jeg3cells, were able to internalise the aPL. Despite treating explants and BeWoswith the same amount of aPL, the level of antibody internalised withinBeWos was less than internalised into the syncytiotrophoblast of explants.Megalin was expressed by BeWos but not Jars or Jeg3 cells. However,BeWos expressed substantially less megalin than the syncytiotrophoblastof placental explants.The internalization of aPL into syncytiotrophoblasts may play an importantrole in regulating the trophoblast death process that leads to the sheddingof syncytial knots, this study adds to our previous data indicating thatmegalin is likely to be the receptor that mediates aPL internalization intotrophoblasts making this pathway a potential therapeutic target.Keywords: antiphospholipid, megalin, internalisation

[P18.04].ANTIPHOSPHOLIPID ANTIBODIES INDUCE ‘‘DEATH’’ AND EXCESSSHEDDING OF SYNCYTIAL KNOTS BY DISRUPTING MITOCHONDRIALFUNCTION

Q Chen*1,2, Y Kang1, B Liu2, P Stone2, L Chamley2. 1The hospital of Obstetrics& Gynaecology, Fudan University, China, 2Department of Obstetrics &Gynaecology, The University of Auckland, New Zealand

Excess trophoblast shedding and deportation is known to be associatedwith preeclampsia a hypertensive disease of pregnancy whose symptomsare preceded by endothelial cell activation. Antiphospholipid antibodies(aPL) are autoantibodies that increase the risk of developing preeclampsianine fold. We have recently found that aPL are internalised into the syn-cytiotrophoblast of placental explants where they subsequently induceincreased shedding of necrotic or aponecrotic trophoblasts that are char-acterised by significantly reduced levels of active caspases 3&7. Thesemore-necrotic trophoblasts can induce the activation of endothelial cells.Here we examine whether aPL alter the trophoblast death process via aneffect on the mitochondria.Placental explants were treated with the monoclonal aPL IIC5 (13ug/ml) orID2 (6ug/ml), or control antibodies, for 2 or 24 hours. Trophoblasts shedfrom the treated explants were collected and contaminating leucocytesremoved. Mitochondrial membrane potential was examined using DiOC2

and confocal microscopy. Cytochrome C levels in shed trophoblasts werequantified by ELISA (R&D).Confocal microscopy showed that mitochondrial function was compro-mised in syncytial knots shed from aPL-treated explants after 2 hours anddid not recover 24 hours post treatment. Significantly higher levels ofcytochrome C (P¼0.001) were released from the mitochondria of syncytialknots that were shed from aPL-treated explants than controls.Our current data, coupled with our previous data showing aPL reduce thelevels of active caspases 3&7 in syncytial knots shed from aPL-treatedexplants, suggest that aPL, once internalised into the syncytiotrophoblast,disrupt normal mitochondrial function in this cell layer which then resultsin aberrant death of portions of the syncytiotrophoblast. The release ofcytochrome C suggests apoptotic death, but the syncytial knots shed formaPL–treated explants are clearly more necrotic than apoptotic as demon-strated by their ability to activate endothelial cells leading us to concludethat aPL induce aponecrotic death in the syncytiotrophoblast.Keywords: antiphospholipid antibodies, mitochondrial, internalisation,cyto chrome C


[P18.05].EFFECT OF siRNA-MEDIATED KNOCKDOWN OF CAVEOLIN-1 ONOXIDATIVE STRESS-INDUCED TROPHOBLAST CELL DEATH

GP Collett*, EA Linton, R Dragovic, CWG Redman, IL Sargent. University ofOxford, United Kingdom

Objectives: Pre-eclampsia is associated with increased shedding of pro-inflammatory placental debris which may occur as a result of exacerbatedoxidative stress-induced cell death in the syncytiotrophoblast. Caveolin-1(cav-1) is expressed in human trophoblast and has been shown to bothpromote and protect from oxidative stress-induced death in other celltypes. Furthermore, in the BeWo human trophoblast cell line hydrogenperoxide (H2O2) treatment is associated with translocation of cav-1 fromthe plasma membrane to the mitochondria, thus potentially affecting theresponse to oxidative stress. In the present study we examined the effect ofcav-1 knockdown on oxidative stress-induced BeWo cell death.Methods: BeWo cells were transfected with a cav-1 siRNA duplex (50nM)or a scrambled, non-silencing control siRNA. After 48h, when cav-1knockdown was confirmed by western blotting, the transfection mediumwas removed and the cells were treated with hydrogen peroxide (0-5mM).Total cell death was assessed by measuring lactate dehydrogenase releaseinto the culture medium, apoptosis was determined by assessing poly(ADP-ribosyl) polymerase cleavage, and necrotic cell death was measuredby monitoring the release of high mobility group box-1 protein (HMGB1)into the culture medium.Results: H2O2 treatment resulted in a dose-dependent increase in BeWocell death. The mode of cell death switched from predominantly apoptoticat low (<1mM) concentrations of H2O2 to predominantly necrotic athigher concentrations. Cav-1 knockdown had no effect on either the extentor mode of cell death at any of the H2O2 concentrations used, comparedwith non-silencing controls. Furthermore, intracellular levels of ATP,which fell rapidly upon H2O2 treatment, were unaffected by cav-1knockdown.Conclusion: Our results suggest that cav-1 has no effect on H2O2-inducedtrophoblast cell death, and therefore may not play a role in the shedding oftrophoblast debris as a result of exacerbated oxidative stress.Keywords: caveolin-1, trophoblast, oxidative stress

[P18.06].NEW DIMENSIONS IN THE QUANTIFICATION AND CHARACTERISATIONOF PLACENTAL AND OTHER CELLULAR MICROPARTICLES ANDNANOPARTICLES IN NORMAL PREGNANCY AND PRE-ECLAMPSIA

RA Dragovic*1, DS Tannetta1, P Harrison2, CWG Redman1, IL Sargent1.1Nuffield Department of Obstetrics & Gynaecology, John Radcliffe Hospital,University of Oxford, United Kingdom, 2Oxford Haemophilia & ThrombosisCentre, Churchill Hospital, Oxford, United Kingdom

Introduction: Pre-eclampsia is associated with altered levels of circulatingparticles derived from the placenta, platelets, immune cells and endo-thelium. These particles are comprised of microparticles (MPs; 100 nm –1 mM) and nanoparticles (exosomes 30 nm – 100 nm). We have previouslymeasured total particles in plasma ultracentrifuge pellets using an ELISA,which does not distinguish between the two populations. We havetherefore investigated the use of a new technology, Nanosight TrackingAnalysis – NTA (which can detect particles in the range of 30 nm - 1 mM) inparallel with flow cytometry to quantify and characterise MP subpopula-tions in pregnancy and pre-eclampsia.Methods: Control syncytiotrophoblast particles (STBMs) were preparedfrom normal caesarean section placentas. Particles were measured in bothplatelet free plasma (PFP) and PFP ultracentrifuge pellets from non-preg-nant (nonP), normal-pregnant (normP) and pre-eclamptic (PET) women,using ELISA, NTA and flow cytometry (BD LSRII digital flow cytometer).Particle count and phenotype was measured by labelling with a novelcombination of cellular particle markers (Lactadherin or Bodipy-mal-eimide) and antibodies to platelet (CD61) and placental particles (NDOG2).Results: Flow cytometric analysis resulted in >90% of STBMs labellingpositive for NDOG2 (detection range 290nm - 1 mM). NanoSight analysisshowed a highly polydisperse distribution (mean size w190 nm). Incomparison to flow cytometry w350 fold more particles were detectedand 85% were <290 nm. Total cellular marker positive particles andcellular marker/CD61 positive particles were significantly increased inboth PFP and the resuspended pellets from nonP vs normP and nonP vs PETsamples. No NDOG2 particles from normP or PET samples were detectedby flow cytometry, although NDOG2 positive particles were detected byELISA and were significantly elevated in PET vs normP samples.Discussion: NTA provides a powerful tool for cellular particle analysis andsuggests the majority of these particles are <290 nm and are therefore toosmall to be detected by flow cytometry.Keywords: Microparticles, Nanoparticles, Pre-eclampsia


[P18.07].DEPTH OF TROPHOBLAST INVASION AND VASCULAR REMODELLING ONDAY 18 AND 21 OF A TRANSGENIC RAT MODEL FOR PRE-ECLAMPSIA

Nele Geusens1, Catherine Luyten1, Lisbeth Vercruysse1, Myriam Hans-sens*1, Robert Pijnenborg1, Ralf Dechend2. 1Dept Obstet & GynaecolK.U.Leuven, Belgium, 2Dept Cardiology Charite University Medicine Berlin,Germany

Background & Objectives: We studied trophoblast invasion and associ-ated uterine spiral artery (SA) remodeling in a transgenic rat model withsymptoms comparable to human pre-eclampsia (PE).Design & Methods: Transgenic Sprague Dawley (SD) rats bearing thehuman angiotensinogen gene and human renin gene were crossed toobtain PE (hAogen \ x hRen _), reversely mated (hAogen _ x hRen \) anda control group consisted ofnormal SP rats . Rats were sacrificed on day 18or day 21 of pregnancy and implantation sites were collected for histologyand immuno stained for cytokeratin (MNF), smooth muscle cell a-actin,the endothelial marker CD31, and fibrinoid (PAS). The mesometrial triangle(MT) is divided into 3 equal parallel layers to evaluate depth of invasion.The KS-400 image analysis system is used to quantify endovasculartrophoblast invasion and associated vascular changes of SA. Statistics areperformed with the Mann-Whitney test.Results: Overall there is, in the entire MT, significantly (P<0.001) moreendovascular trophoblast overlying the SA lumen contours in the PE groupcompared to that observed in the control and RM group. This is particularlytrue in the deeper layers. Yet, control rats seem to catch up with the PE ratsby day 21. Inspite of the deeper endovascular trophoblast invasion in thePE rats, vascular smooth muscle cell remodelling was less pronounced inthat group as compared to that observed in the reversely mated and thecontrol rats both at 18 and 21 days of pregnancy.Conclusion: The findings of more endovascular trophoblast with lesspronounced smooth muscle cell remodelling in the SA of the PE group arequite different from observations in human PE where less endovasculartrophoblast and almost absent remodelling of the myometrial segment ofthe SA is seen.Keywords: trophoblast invasion, remodellling spiral arteries, transgenicrat model, pre-eclampsia

[P18.08].P53 MEDIATED APOPTOSIS IN A MODEL OF HUMAN PLACENTALVILLOUS FUNCTION

A.N. Sharp, A.E.P. Heazell, P.N. Baker, I.P. Crocker*. University of Man-chester, United Kingdom

Introduction: The placental conditions of pre-eclampsia and intrauterinegrowth restriction are characterised by altered villous cell turnover andexaggerated expression of the cell regulator p53, implying a role introphoblast apoptosis. In BeWo cells stimulation of the p53 pathway canbe achieved with the small molecule activator, Nutlin-3, whilst p53 activitycan be inhibited with a second compound, Pifithrin-a, which may correctthese aberrant apoptotic events. The effects of these compounds weretested in vitro on placental villous fragments.Methods: Third trimester placental explants from uncomplicated preg-nancies were cultured at 6%O2 in the presence of Nutlin-3 alone or incombination with Pifithrin-a. Culture medium was collected for assess-ment of lactate dehydrogenase (necrosis) and human chorionic gonado-trophin (hCG) secretion (trophoblast differentiation). Tissue was formalinfixed and wax embedded for immunohistochemistry and quantitative PCRperformed for mRNA expression. Trophoblast apoptosis was assessed byM30 staining and reported in proportion to villous area.Results: Nutlin-3 increased trophoblast apoptosis in cultured explants(control vs. 40mM Nutlin, 0.75�0.11 median (SD) vs. 3.52�0.38, p<0.01,Kruskal-Wallis, n¼5) with no effect on LDH or hCG release. Nutlin-3treatment increased p21 gene expression (29.4�35.7 vs. 4.66�2.49,p<0.01, Mann-Whitney Test), but not p53 or Mdm2. Dual treatment withNutlin-3 and Pifithrin-a (5mM) returned apoptotic levels to control values(0.84�0.40 vs. 1.48�0.50, ns, Kruskal-Wallis, n¼5), suppressing Nutlin-3induced apoptosis of trophoblast by 58%.Discussion: Nutlin-3 modulates p53 in third trimester placental tropho-blasts, stimulating apoptosis rather than necrosis. Pifithrin-a can be usedto restrict this pathway, potentially offering a way of regulating excessiveplacental apoptosis. Further experiments will establish a role for thesecompounds in complicated pregnancies in vivo.Keywords: pre-eclampsia, p53, apoptosis, trophoblast


[P19.01].EXPRESSION OF ANGIOGENIC FACTORS IN PLACENTA OF STRESSEDRATS

Isis Paloppi Correa1, Rodrigo Ruano1, Nilton Hideto Takiuti1, Salet Terezinhade Souza1, Estela Bevilacqua2, Marcelo Zugaib*1. 1Obstetrics Department,Faculty of Medicine, University of Sao Paulo, Brazil, 2Institute of BiomedicalSciences, University of Sao Paulo, Brazil

The aim of present study was to analyze the influence of the stress inpregnant rats on gestational parameters, on the placental morphology andon the placental gene expression of VEGF, PlGF and, its receptors. Theseparameters were evaluated on gestation day 20 after chronic and acutestress protocols during gestation. Fetal and placental weights werestatistically smaller in the stressed animals comparing to controls.Morphologically the placentas exhibited reduction in the junctional zoneand degenerative cells. Although VEGF, PlGF, VEGFR1 and VEGFR2 wereexpressed in all placentas, stress condition significantly changed theplacental gene expression in comparison to control group, increasingVEGFA (p<0.05) and, reducing PlGF, VEGFR-1 and VEGFR-2 expression(p>0.05). In conclusion, the present study showed that in the stresspregnant group, placental and fetal growth restriction is observed, sug-gesting abnormalities in the gestational physiology. These animals showedpersistent hypertension which suggests a hypotensive response such asthose induced by VEGF; consistent with that, placental VEGF wasincreased. However, because at the same sites PlGF and receptors VEGFR1/R2 expressions were relevantly reduced, it is possible that the VEGF actionon stress-induced hypertension has been limited. This model of investi-gation shows at least one mechanism by which stress can act on theplacental physiology and, may further help us understand stress-inducedgestational disorders.Financial support: FAPESP and CNPq.Keywords: Stress, VEGF, PlGF, VEGFR

[P19.02].MATRIX METALLOPROTEINASE-12, AN IMPORTANT MEDIATOR OFELASTOLYSIS IN REMODELLING HUMAN SPIRAL ARTERIES?

LK Harris*1, PN Baker1, JE Cartwright2, GS Whitley2, V Dive3, A Yiotakis4.1University of Manchester, United Kingdom, 2St George’s University ofLondon, United Kingdom, 3Institut de Biologie et de Technologies deSalcay, France, 4University of Athens, Greece

Introduction: Remodelling of the uterine spiral arteries involves degra-dation of elastin fibres within the arterial media to facilitate a permanentincrease in vessel diameter; however, the mediator(s) of elastolysis arecurrently unknown. We have previously shown that first trimester cyto-trophoblasts (CTB) and vascular smooth muscle cells (SMC) utilize matrixmetalloproteinases (MMP) to degrade elastin in vitro. As SMC and CTBexpress high levels of the elastolytic enzyme MMP-12, we hypothesizedthat these cells employ MMP-12 to catabolise elastin.Methods: CTB were isolated from first trimester placenta by trypsindigestion and density gradient centrifugation. Triton-X100 extracts of CTBand SMC were incubated with the elastase substrate N-succinyl-(L-alanine)3-p-nitroanilide, in the presence of a broad spectrum MMPinhibitor or a specific inhibitor of MMP-12. Elastase activity was calculatedfrom a standard curve prepared using porcine pancreatic elastase. CTBcultured with elastin in the presence or absence of MMP inhibitors werestained with an antibody to cytokeratin-7, and the number of cellsengulfing elastin fibres was quantified.Results: Triton-X100 extracts of SMC and CTB exhibited MMP-dependentelastase activity, consistent with the presence of membrane-associatedelastases. A specific inhibitor of MMP-12 reduced elastolysis to 23.3�8.7%of control activity in SMC and 31.7�10.9% in CTB, compared to a broadspectrum MMP inhibitor which reduced activity to 2.65�2.7% in SMC and0.13�0.1% in CTB. Inhibition of MMP activity did not reduce the percent ofcells containing intracellular elastin fragments.Discussion: These data implicate MMP-12 as a primary mediator of elas-tolysis in first trimester CTB and SMC, but demonstrate that MMP-12 is notrequired for elastin engulfment. As we have previously shown that MMP-12 expression is induced in the media of excised spiral arteries culturedwith CTB-conditioned medium, we hypothesise that the coordinatedactions of invasive trophoblast and spiral artery SMC mediate local elastincatabolism during vessel transformation.Keywords: trophoblast invasion, MMP-12, elastin, arterial remodelling


[P19.03].LEUKOCYTE MEDIATED MECHANISMS OF VASCULAR REMODELINGIN VITRO

A D Hazan*1,2, S D Smith3,4, R L Jones3,4, S J Lye1,2, C E Dunk2. 1University ofToronto, Canada, 2Samuel Lunenfeld Research Institute, Mount SinaiHospital, Canada, 3University of Manchester, United Kingdom, 4St. Mary’sHospital, United Kingdom

Introduction: While there is an abundant population of immune cells inthe decidua, their functional role in vascular transformation has yet to befully established. In this study we investigate the association of uterineNatural Killer cells (uNK) and macrophages with vascular remodelingduring early gestation and the potential mechanisms by which theyregulate these processes.Methods: Patient-matched first trimester (6-9 weeks) placental anddecidual tissues are collected. Decidual explants are cultured alone (n¼13)or with placenta (n¼13) over 6 days. Placenta-induced vascular trans-formation and leukocyte localization in these decidua are assessed byimmunohistochemistry and quantitative image analysis at increasingdepths from the site of placental contact (0-500, 500-1000, & 1000-2000mm) and 15mm radiating distances from vessel lumens.Results: Vascular transformation is indicated by a decrease (p<0.05) in‘smooth muscle actin/vessel lumen’ area in the proximal 1000mm of co-cultures compared to decidual controls. In actively remodeling vessels atday 3 there is an increase (p<0.0003) in the number of uNK and macro-phages within 15mm of the vessel lumen. Farther from the vessel lumen(15-30mm) there is an increase in macrophages (p<0.01). Concurrentlythere is disruption of the vascular smooth muscle cell (vsmc) layers,separation and migration of individual vsmc, and gradual loss of smoothmuscle actin staining, suggesting dedifferentiation of vsmc. We alsoobserve matrix metalloproteinase(MMP)-9 expression by uNKs andvascular cells. Inhibition of MMP-2/9 results in similar ‘smooth muscle/lumen’ ratios between co-cultures and controls. TUNEL staining demon-strates that vsmc and endothelium are apoptotic in remodeling vessels anddual immunofluorescence using lysozyme muramidase indicates macro-phage-mediated phagocytosis.Discussion: These experiments identify, for the first time, potentialmechanisms involved in decidual vascular remodeling induced by thepresence of placenta. Temporal differences in macrophage and uNKassociation with vessels during remodeling indicate distinct functionalroles for these cells in a tightly regulated process.Keywords: leukocytes, decidua, trophoblast, vascular remodeling

[P19.04].NEW ROUTES OF TROPHOBLAST INVASION? INVESTIGATIONS WITHA CO-CULTURE CONFRONTATION MODEL SYSTEM

G Moser*, K Orendi, M Gauster, M Siwetz, N Flieser, B Huppertz. MedicalUniversity Graz, Austria

Objectives: Secretory products delivered by the uterine glands into theintervillous space prior to the establishment of maternal blood flow into theplacenta are considered to play an important role in the nutrition ofthe embryo (Burton et al. 2002). However, these authors did not provide anyidea of how such secretory products may reach the intervillous space.Therefore, we tested putative new routes of trophoblast invasionwith doubletissue co-culture in-vitro confrontation models for trophoblast invasion.Methods: Pieces of decidua parietalis (6-10 weeks gestation) were con-fronted directly with villous explants from the same pregnancy (directconfrontation – without epithelium) or after separate preculture for 72h(indirect confrontation – with epithelium). For ‘‘indirect confrontation’’decidua pieces re-epithelialized during preculture under constant agitation.All confrontations were harvested after 72h, cryosectioned and processedfor immunohistochemistry and/or double labeling immunofluorescenceusing antibodies against cytokeratin 7, HLA-G and entactin.Results: In both models (direct and indirect confrontation) confrontationof villous and decidual tissues resulted in formation of trophoblastic cellcolumns and their adhesion to decidual tissues. Immunohistochemicalstaining with antibodies against HLA-G showed invasion of extravilloustrophoblast (EVT) into decidual tissues. Immunofluorescent doublelabeling against either HLA-G/entactin or cytokeratin 7/entactin showedthat EVT invaded through the decidual stroma and along the basal laminaof decidual epithelium and glands. Thus, during invasion along the basallamina of the epithelium EVT reached glands and single trophoblasts werealso found in the lumen of glands.Conclusions: Enders et al. (1983) showed that in the rhesus monkeyextravillous trophoblast extended along the basal lamina of the uterineepithelium and into the neck of adjacent uterine glands. Our data showthat extravillous trophoblast in the human may follow similar routes, so farnot described yet. They may reach and open uterine glands to facilitatehistiotrophic embryo nutrition.Keywords: uterine glands, confrontation model, extravillous trophoblasts,histiotrophic nutrition


[P19.05].MECHANISM OF PROPROTEIN CONVERTASE 6 ACTION DURINGDECIDUALIZATION: REGULATION OF BONE MORPHOGENETICPROTEIN 2 ACTIVATION

S Heng, B Hardman, S Paule, H Singh, L Salamonsen, G Nie*. Prince Henry’sInstitute of Medical Research, Melbourne, Australia

Introduction: Proprotein convertase 5/6 (PC6), a member of the propro-tein convertase (PC) family, is a critical endometrial factor for implanta-tion. PC6 is up-regulated in the endometrium specifically at implantationin association with epithelial differentiation (in human and monkey) andstromal cell decidualization (in the mouse, human and monkey). Knock-down of PC6 inhibits decidualization. PCs function by converting a range ofimportant precursor proteins into their bioactive forms. One group of suchproteins are the transforming growth factor beta (TGF-beta) superfamilyproteins. They are first synthesized as larger biologically inactive precur-sors, and then are processed by PCs into their active forms. Bonemorphogenetic protein 2 (BMP2) is a TGF-beta superfamily member anddemonstrated to be essential for decidualization. We hypothesized thatBMP2 is one of the proteins that PC6 activates during decidualization.Methods: Freshly isolated stromal cells from human endometrium weredecidualized in culture with and without inhibition of PC6 activity. Thefull-length (precursor, non-active) and processed (activated) forms ofBMP2 were determined in cellular lysates and media. The proteolyticprocessing of BMP2 by PC6 was confirmed in vitro. To further confirm therole of PC6 in activating BMP2 in decidualization, active BMP2 was addedinto cells to rescue decidualization arrest caused by PC6 inhibition.Results: PC6 was the only PC member that was significantly up-regulatedduring decidualization. The precursor form of BMP2 was reduced whereas theactive form was increased during decidualization. Inhibition of PC6 activityinhibited decidualization, and this inhibition was accompanied by a totalinhibition of the production of active BMP2. Addition of recombinant activeBMP2 partially rescued the decidualization arrest caused by PC6 inhibition.Discussion: Both PC6 and BMP2 are known to be critical for decidualiza-tion. This study demonstrated that PC6 regulates decidualization by acti-vating molecules such as BMP2 that are essential for decidualization.Keywords: PC6, BMP2, Decidualization, Activation

[P19.07].IDENTIFICATION OF CHEMOKINES INVOLVED IN RECRUITMENT OFDECIDUAL LEUKOCYTES TO REMODELLING SPIRAL ARTERIES

SD Smith*1, JD Aplin1, LK Harris1, CE Dunk1,2, RL Jones1. 1University ofManchester, United Kingdom, 2University of Toronto, Canada

Introduction: Remodelling of uterine spiral arteries in the first 20 weeksof gestation is essential for a healthy pregnancy. We have recently shownthat macrophages and uNK cells infiltrate the wall of remodelling decidualspiral arteries prior to extravillous trophoblast (EVT) colonisation,providing evidence for a role for decidual leukocytes in the early stages ofremodelling. However, leukocytes do not infiltrate the wall of arteriesdistant from the implantation site (decidua parietalis) suggesting a localtrigger for leukocyte recruitment. We hypothesized that trophoblast-secreted factors activate spiral arteries to produce chemokines, whichchemoattract leukocytes into remodelling vessels.Methods: Decidual endothelial cells were isolated from first trimesterdecidua (8-12 weeks gestation). Vascular smooth muscle cells (VSMC,derived from human aorta) and decidual endothelial cells were treatedwith EVT-conditioned medium generated from villous tip outgrowths.Chemokine expression by treated and untreated VSMC and endothelialcells was analysed using an unbiased PCR genearray approach, to examineexpression of 80 chemokines/cytokines/receptors. Immunohistochemistryfor selected chemokines in decidua basalis (8-12 weeks gestation) wasperformed to validate expression patterns in vivo.Results: 40 genes were differentially regulated in both VSMC and decidualendothelial cells in response to EVT conditioned medium. Of particularinterest were macrophage chemoattractants: CCL5, CCL7, CCL3 and CCL4,that were upregulated between 2-36 fold in treated compared to control inboth cell types. Furthermore, uNK chemoattractants CXCL12 and CX3CL1were upregulated in VSMC and endothelial cells respectively. Immuno-histochemistry of decidua basalis confirmed CXCL12, CCL4 and CX3CL1localisation to spiral artery VSMC and endothelial cells.Discussion: These data suggest that factors secreted by EVT promotechemokine expression by vascular cells. We have identified chemo-attractants released by vascular cells that may recruit macrophages (CCL5,CCL7, CCL3 and CCL4) and uNK cells (CXCL12 and CX3CL1) to spiral arteries,where they participate in the early stages of remodelling.Keywords: Decidua, Leukocytes, Trophoblast, Chemokines


[P19.08].PRODUCTION OF ANGIOPOIETIN-2 BY DECIDUAL ENDOTHELIAL CELLSIN HYPOXIA, AND IN FIRST VERSUS THIRD TRIMESTER MATERNALSERUM

CA Woolnough*1,2, Y Wang2, V Tasevski1,2, E Gallery2, J Morris2.1Fetal Maternal Medicine (PaLMS), Royal North Shore Hospital, Australia,2Perinatal Research, Kolling Institute, Royal North Shore Hospital, Australia

Introduction: During pregnancy the placenta, fetus and uterus undergoangiogenesis which is accompanied by a rise in Angiopoietin-2 (Ang-2)levels in maternal serum peaking in the first trimester and graduallydeclining throughout gestation. The first objective of this study was todetermine whether decidual endothelial cells (DECS) may be the source ofthe Ang-2 observed in maternal serum. The second objective was toinvestigate potential factors which may regulate Ang-2 production byDECS (1) first compared to third trimester maternal serum and (2) hypoxia.Methods: DECS (n¼8) were isolated from maternal decidua obtained atcaesarean section clinically following normal pregnancies, and cultured untilpassage five. DECS were exposed to hypoxia (1% and 3%) and normoxia. In eachoxygen condition, DECS were cultured in first trimester and third trimesterserum. DEC supernatants were collected and assayed for Ang-2 using ELISA,and Ang-2 in cellular lysates was measured by western blotting. Cellular RNAwas extracted for reverse transcriptase PCR and Ang-2 semi-quantification.Results: Ang-2 was detected in DEC supernatant, cellular protein andcellular RNA. DECS released statistically significantly more Ang-2 whencultured in first compared to third trimester serum, and when cultured in1% compared with 3% hypoxia and normoxia (Figure 1). Cellular Ang-2

levels followed similar patterns however Ang-2 RNA levels were notsignificantly different across the culturing conditions.

Figure 1. Ang-2 released by DECS under different culturing conditions *p<0.05 Ang-2levels are higher in first compared to third trimester serum, D p<0.05 Ang-2 levels arehigher in 1% hypoxia compared to normoxia.

Discussion: DECS release substantial amounts of Ang-2 indicating thatthese cells are a source of the Ang-2 seen in maternal serum. Proteinlevels of Ang-2 are higher when DECS are cultured in first trimesterserum and in 1% hypoxia indicating that serum factors in addition toa hypoxic environment may trigger DECS to produce more Ang-2 in thefirst trimester. There were no significant differences in Ang-2 RNA levelsacross the different treatments indicating that DEC Ang-2 is controlled attranslation.Keywords: Angiopoietin-2, Hypoxia, Decidua, Endothelial cells

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